Tendon-inducing compositions

ABSTRACT

Compositions of proteins with tendon/ligament-like tissue inducing activity are disclosed. The compositions are useful in the treatment of tendinitis and tendon or ligament defects and in related tissue repair.

RELATED APPLICATIONS

The instant application is a division of Ser. No. 08/362,670, filed Dec.22, 1994, now U.S. Pat. No. 5,658,882, which is a continuation-in-partof 08/217,780, filed Mar. 25, 1994, presently abandoned, 08/164,103,filed Dec. 7, 1993, presently abandoned and 08/333,576, filed Nov. 2,1994, now U.S. Pat. No. 6,027,919.

FIELD OF THE INVENTION

The present invention relates to a novel family of purified proteins,and compositions containing such proteins, which compositions are usefulfor the induction of tendon/ligament-like tissue formation, woundhealing and ligament and other tissue repair. These proteins may also beused in compositions for augmenting the activity of bone morphogeneticproteins.

BACKGROUND OF THE INVENTION

The search for the molecule or molecules responsible for formation ofbone, cartilage, tendon and other tissues present in bone and othertissue extracts has led to the discovery of a novel set of moleculescalled the Bone Morphogenetic Proteins (BMPs). The structures of severalproteins, designated BMP-1 through BMP-11, have previously beenelucidated. The unique inductive activities of these proteins, alongwith their presence in bone, suggests that they are important regulatorsof bone repair processes, and may be involved in the normal maintenanceof bone tissue. There is a need to identify additional proteins whichplay a role in forming other vital tissues. The present inventionrelates to the identification of a family of proteins, which havetendon/ligament-like tissue inducing activity, and which are useful incompositions for the induction of tendon/ligament-like tissue formationand repair.

SUMMARY OF THE INVENTION

In one embodiment, the present invention comprises DNA moleculesencoding a tendon/ligament-like inducing protein which the inventorshave named V1-1. This novel protein is now called BMP-12. The presentinvention also includes DNA molecules encoding BMP-12 related proteins.BMP-12 related proteins are a subset of the BMP/TGF-β/Vg-1 family ofproteins, including BMP-12 and VL-1, which are defined astendon/ligament-like tissue inducing proteins encoded by DNA sequenceswhich are cloned and identified, e.g., using PCR, using BMP-12 specificprimers, such as primers #6 and #7 described below, with reducedstringency conditions. It is preferred that the DNA sequences encodingBMP-12 related proteins share at least about 80% homology at the aminoacid level from amino acids with amino acids #3 to #103 of SEQ ID NO:1.

The DNA molecules preferably have a DNA sequence encoding the BMP-12protein, the sequence of which is provided in SEQ ID NO:1, or a BMP-12related protein as further described herein. Both the BMP-12 protein andBMP-12 related proteins are characterized by the ability to induce theformation of tendon/ligament-like tissue in the assay described in theexamples.

The DNA molecules of the invention preferably comprise a DNA sequence,as described in SEQUENCE ID NO:1; more preferably nucleotides #496 to#882, #571 to #882 or #577 to #882 of SEQ ID NO:1; or DNA sequenceswhich hybridize to the above under stringent hybridization conditionsand encode a protein which exhibits the ability to formtendon/ligament-like tissue. The DNA molecules of the invention may alsocomprise a DNA sequence as described in SEQ ID NO:25; more preferablynucleotides #604 or #658 to #964 of SEQ ID NO:25.

The DNA molecules of the invention also include DNA molecules comprisinga DNA sequence encoding a BMP-12 related protein with the amino acidsequence shown in SEQ ID NO:2 or SEQ ID NO:26, as well as naturallyoccurring allelic sequences and equivalent degenerative codon sequencesof SEQ ID NO:2 or SEQ ID NO:26. Preferably, the DNA sequence of thepresent invention encodes amino acids #-25 to #104, #1 to #104 or #3 to#103 of SEQ ID NO:2; or amino to #120 or #19 to #120 of SEQ ID NO:26.The DNA sequence may comprise, in a 5′ to 3′ direction, nucleotidesencoding a propeptide, and nucleotides encoding for amino acids #-25 to#104, #1 to #104 or #3 to #103 of SEQ ID NO:2; or amino acids #1 to #120or #19 to #120 of SEQ ID NO:26. The propeptide useful in the aboveembodiment is preferably selected from the group consisting of nativeBMP-12 propeptide and a protein propeptide from a different member ofthe TGF-B superfamily or BMP family. The invention further comprises DNAsequences which hybridize to the above DNA sequences under stringenthybridization conditions and encode a BMP-12 related protein whichexhibits the ability to induce formation of tendon/ligament-like tissue.

In other embodiments, the present invention comprises host cells andvectors which comprise a DNA molecule encoding the BMP-12 protein, or aBMP-12 related protein. The host cells and vectors may further comprisethe coding sequence in operative association with an expression controlsequence therefor.

In another embodiment, the present invention comprises a method forproducing a purified BMP-12 related protein, said method comprising thesteps of culturing a host cell transformed with the above DNA moleculeor vector comprising a nucleotide sequence encoding a BMP-12 relatedprotein; and (b) recovering and purifying said BMP-12 related proteinfrom the culture medium. In a preferred embodiment, the method comprises(a) culturing a cell transformed with a DNA molecule comprising thenucleotide sequence from nucleotide #496, #571 or #577 to #879 or #882as shown in SEQ ID NO:1; or the nucleotide sequence from #604 or #658 to#963 of SEQ ID NO:25; and

(b) recovering and purifying from said culture medium a proteincomprising the amino acid sequence from amino acid #−25, #1 or #3 toamino acid #103 or #104 as shown in SEQ ID NO:2; or from amino acid #1or #19 to amino acid #120 as shown in SEQ ID NO:26. The presentinvention also includes a purified protein produced by the abovemethods.

The present invention further comprises purified BMP-12 related proteincharacterized by the ability to induce the formation oftendon/ligament-like tissue. The BMP-12 related polypeptides preferablycomprise an amino acid sequence as shown in SEQ ID NO:2. The polypeptidemore preferably comprise amino acids #−25, #1 or #3 to #103 or #104 asset forth in SEQ ID NO:2; or amino acids #1 or #19 to #120 as set forthin SEQ ID NO:26. In a preferred embodiment, the purified polypeptide maybe in the form of a dimer comprised of two subunits, each with the aminoacid sequence of SEQ ID NO:2.

In another embodiment, the present invention comprises compositionscomprising an effective amount of the above-described BMP-12 relatedproteins. In the compositions, the protein may be admixed with apharmaceutically acceptable vehicle.

The invention also includes methods for tendon/ligament-like tissuehealing and tissue repair, for treating tendinitis, or other tendon orligament defects, and for inducing tendon/ligament-like tissue formationin a patient in need of same, comprising administering to said patientan effective amount of the above composition.

Other embodiments include chimeric DNA molecules comprising a DNAsequence encoding a propeptide from a member of the TGF-β superfamily ofproteins linked in correct reading frame to a DNA sequence encoding aBMP-12 related polypeptide. One suitable propeptide is the propeptidefrom BMP-2. The invention also includes heterodimeric protein moleculescomprising one monomer having the amino acid sequence shown in SEQ IDNO:2, and one monomer having the amino acid sequence of another proteinof the TGF-β subfamily.

Finally, the present invention comprises methods for inducingtendon/ligament-like tissue formation in a patient in need of samecomprising administering to said patient an effective amount of acomposition comprising a protein which exhibits the ability to induceformation of tendon/ligament-like tissue, said protein having an aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:26. Theamino acid sequences are more preferably one of the following: (a) aminoacids #−25, #1 or #3 to #103 or #104 of SEQ ID NO:2; (b) amino acids #1or #19 to #119 or #120 of SEQ ID NO:4; (c) amino acids #1 or #19 to #119or #120 of SEQ ID NO:26; (d) mutants and/or variants of (a), (b) or (c)which exhibit the ability to form tendon and/or ligament. In otherembodiments of the above method, the protein is encoded by a DNAsequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:25, more preferablyone of the following: (a) nucleotides #496, #571 or #577 to #879 or #882of SEQ ID NO:1; (b) nucleotides #845 or #899 to #1201 or #1204 of SEQ IDNO:3; (c) nucleotides #605 or #659 to #961 or #964 of SEQ ID NO:25; and(d) sequences which hybridize to (a) or (b) under stringenthybridization conditions and encode a protein which exhibits the abilityto form tendon/ligament-like tissue.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence encoding the human BMP-12.

SEQ ID NO:2 is the amino acid sequence comprising the mature humanBMP-12 polypeptide.

SEQ ID NO:3 is the nucleotide sequence encoding the protein MP52.

SEQ ID NO:4 is the amino acid sequence comprising the mature MP52polypeptide.

SEQ ID NO:5 is the nucleotide sequence of a specifically amplifiedportion of the human BMP-12 encoding sequence.

SEQ ID NO:6 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:5.

SEQ ID NO:7 is the nucleotide sequence of a specifically amplifiedportion of the human VL-1 encoding sequence.

SEQ ID NO:8 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:7.

SEQ ID NO:9 is the nucleotide sequence of the plasmid pALV1-781, usedfor expression of BMP-12 in E. coli.

SEQ ID NO:10 is the nucleotide sequence of a fragment of the murineclone, mV1.

SEQ ID NO:11 is the amino acid sequence of a fragment of the murineprotein encoded by mV1.

SEQ ID NO:12 is the nucleotide sequence of a fragment of the murineclone, mV2.

SEQ ID NO:13 is the amino acid sequence of a fragment of the murineprotein encoded by mV2.

SEQ ID NO:14 is the nucleotide sequence of a fragment of the murineclone, mV9.

SEQ ID NO:15 is the amino acid sequence of a fragment of the murineprotein encoded by mV9.

SEQ ID NO:16 is the amino acid sequence of a BMP/TGF-β/Vg-1 proteincorisensus sequence. The first Xaa represents either Gln or Asn; thesecond Xaa represents either Val or Ile.

SEQ ID NO:17 is the nucleotide sequence of oligonucleotide #1.

SEQ ID NO:18 is the amino acid sequence of a BMP/TGF-β/Vg-1 proteinconsensus sequence. The Xaa represents either Val or Leu.

SEQ ID NO:19 is the nucleotide sequence of oligonucleotide #2.

SEQ ID NO:20 is the nucleotide sequence of oligonucleotide #3.

SEQ ID NO:21 is the nucleotide sequence of oligonucleotide #4.

SEQ ID NO:22 is the nucleotide sequence of oligonucleotide #5.

SEQ ID NO:23 is the nucleotide sequence of oligonucleotide #6.

SEQ ID NO:24 is the nucleotide sequence of oligonucleotide #7.

SEQ ID NO:25 is the nucleotide sequence of the human VL-1 (BMP-13)encoding sequence.

SEQ ID NO:26 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:25.

SEQ ID NO:27 is the nucleotide sequence encoding a fusion of BMP-2propeptide and the mature coding sequence of BMP-12.

SEQ ID NO:28 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:27.

SEQ ID NO:29 is the nucleotide sequence encoding the murine mV1 protein.the first Xaa is Val, Ala, Glu or Gly; the second Xaa is Ser, Pro Thr orAla; the third Xaa is Ser or Arg; the fourth Xaa is Leu, Pro, Gin orArg; the fifth Xaa is Cys or Trp; the sisth Xaa is Val, Ala, Asp or Gly;the seventh Xaa is Val, Ala, Glu or Gly; the eighth Xaa is Gln, Lys orGlu.

SEQ ID NO:30 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:29. The first Xaa through the eighth Xaa are thesame as in SEQ ID NO:29.

SEQ ID NO:31 is the nucleotide sequence encoding the murine mV2 protein.the first Xaa is Pro or Thr; the second Xaa is Val.

SEQ ID NO:32 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:31. Xaa and the second Xaa are the same as in SEQID NO:31.

SEQ ID NO:33 is the nucleotide sequence encoding human BMP-12 protein.

SEQ ID NO:34 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:33.

SEQ ID NO:35 is the nucleotide sequence of oligonucleotide #8.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a comparison of the human BMP-12 and human MP52 sequences. Thesequence of human BMP-12 is set forth in SEQ ID NO:1. The sequence ofhuman MP52 is set forth in SEQ ID NO;3.

DETAILED DESCRIPTION OF THE INVENTION

The DNA sequences of the present invention are useful for producingproteins which induce the formation of tendon/ligament-like tissue, asdescribed further below. The DNA sequences of the present invention arefurther useful for isolating and cloning further DNA sequences encodingBMP-12 related proteins with similar activity. These BMP-12 relatedproteins may be homologues from other species, or may be relatedproteins within the same species.

Still, a further aspect of the invention are DNA sequences coding forexpression of a tendon/ligament-like tissue inducing protein. Suchsequences include the sequence of nucleotides in a 5′ to 3′ directionillustrated in SEQ ID NO:1 or SEQ ID NO:25, DNA sequences which, but forthe degeneracy of the genetic code, are identical to the DNA sequenceSEQ ID NO:1 or 25, and encode the protein of SEQ ID NO:2 or 26. Furtherincluded in the present invention are DNA sequences which hybridizeunder stringent conditions with the DNA sequence of SEQ ID NO:1 or 25and encode a protein having the ability to induce the formation oftendon or ligament. Preferred DNA sequences include those whichhybridize under stringent conditions as described in Maniatis et al,Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory(1982), pages 387 to 389. Finally, allelic or other variations of thesequences of SEQ ID NO:1 or 25, whether such nucleotide changes resultin changes in the peptide sequence or not, but where the peptidesequence still has tendon/ligament-like tissue inducing activity, arealso included in the present invention.

The human BMP-12 DNA sequence (SEQ ID NO:1) and amino acid sequence (SEQID NO:2) are set forth in the Sequence Listings. Another protein that isuseful for the compositions and methods of the present invention isVL- 1. VL- 1 is a BMP-12 related protein which was cloned usingsequences from BMP-12. The inventors have now designated VL-1 as BMP-13.A partial DNA sequence of VL-1 (SEQ ID NO:7) and the encoded amino acidsequence (SEQ ID NO:8); as well as a DNA sequence encoding the matureVL-1 (SEQ ID NO:25) and the encoded amino acid sequence (SEQ ID NO:26)are set forth in the Sequence Listings. Although further descriptionsare made with reference to the BMP-12 sequence of SEQ ID NO:1 and 2, itwill be recognized that the invention includes similar modifications andimprovements which may be made to other BMP-12 related sequences, suchas the VL-1 sequence shown in SEQ ID NO:25 and 26.

The sequence of BMP-12 shown in SEQ ID NO:1 includes the entire maturesequence and approximately 190 amino acids of the propeptide. The codingsequence of the mature human BMP-12 protein appears to begin atnucleotide #496 or #571 and continues through nucleotide #882 of SEQ IDNO:1. The first cysteine in the seven cysteine structure characteristicof TGF-β proteins begins at nucleotide #577. The last cysteine ends at#879. Thus, it is expected that DNA sequences encoding active BMP-12species will comprise nucleotides #577 to #879 of SEQ ID NO:1.

It is expected that BMP-12, as expressed by mammalian cells such as CHOcells, exists as a heterogeneous population of active species of BMP-12protein with varying N-termini. It is expected that all active specieswill contain the amino acid sequence beginning with the cysteine residueat amino acid #3 of SEQ ID NO:2 and continue through at least thecysteine residue at amino acid 103 or until the stop codon after aminoacid 104. Other active species contain additional amino acid sequence inthe N-terminal direction. As described further herein, the N-termini ofactive species produced by mammalian cells are expected to begin afterthe occurrence of a consensus cleavage site, encoding a peptide sequenceArg-X-X-Arg (SEQ ID NO:2, residues −4 to −1). Thus, it is expected thatDNA sequences encoding active BMP-12 proteins will have a nucleotidesequence comprising the nucleotide sequence beginning at any ofnucleotides #196, 199, 208, 217, 361, 388, 493, 496 or 571 to nucleotide#879 or 882 of SEQ ID NO:1.

The N-terminus of one active species of human BMP-12 has beenexperimentally determined by expression in E. coli to be as follows:[M]SRXSRKPLHVDF (SEQ ID NO:2 residues 1 to 12) wherein X designates anamino acid residue with no clear signal, which is consistent with acysteine residue at that location. Thus, it appears that the N-terminusof this species of BMP-12 is at amino acid #1 of SEQ ID NO:1, and a DNAsequence encoding said species of BMP-12 would start at nucleotide #571of SEQ ID NO:1. The apparent molecular weight of this species of humanBMP-12 dimer was determined by SDS-PAGE to be approximately 20-22 kd ona Novex 16% tricine gel. The pI of this molecule is approximately 4.9.The human BMP-12 protein exists as a clear, colorless solution in 0.1 %trifluoroacetic acid. The N-terminus of another active species of humanBMP-12 has been experimentally determined by expression in E. coli to be[M]TALA (SEQ ID NO:2, residues −25 to −12). The pI of this molecule isapproximately 7.0. The apparent molecular weight of this species ofhuman BMP-12 dimer was determined by SDS-PAGE to be approximately 25-27kd on a Novex 16% tricine gel. The human BMP-12 protein exists as aclear, colorless solution in 0.1% trifluoroacetic acid.

As described earlier, BMP-12 related proteins are a subset of theBMP/TGF-β/Vg-1 family of proteins, including BMP-12 and VL-1, which canbe defined as tendon/ligament-like tissue inducing proteins encoded byDNA sequences which can be cloned and identified, e.g., using PCR, usingBMP-12 specific primers, such as primers #6 and #7 described below, withreduced stringency conditions. It is preferred that DNA sequences of thepresent invention share at least about 80% homology at the amino acidlevel from amino acids with the DNA encoding amino acids #3 to #103 ofSEQ ID NO:1. For the purposes of the present invention, the term BMP-12related proteins does not include the human MP52 protein. Using thesequence information of SEQ ID NO:1 and SEQ ID NO:3, and the comparisonprovided in FIG. 1, it is within the skill of the art to design primersto the BMP-12 sequence which will allow for the cloning of genesencoding BMP-12 related proteins.

One example of the BMP-12-related proteins of the present invention isVL-1, presently referred to as BMP-13. The sequence of the full matureBMP-13 sequence and at least a part of the propeptide of BMP-13 is givenin SEQ ID NO:25. Like BMP-12, it is expected that BMP-13, as expressedby mammalian cells such as CHO cells, exists as a heterogeneouspopulation of active species of BMP-13 protein with varying N-termini.It is expected that all active species will contain the amino acidsequence beginning with the cysteine residue at amino acid #19 of SEQ IDNO:26 and continue through at least the cysteine residue at amino acid119 or until the stop codon after amino acid 120. Other active speciescontain additional amino acid sequence in the N-terminal direction. Asdescribed further herein, the N-termini of active species produced bymammalian cells are expected to begin after the occurrence of aconsensus cleavage site, encoding a peptide sequence Arg-X-X-Arg (SEQ IDNO:26, residues −4 to −1). Thus, it is expected that DNA sequencesencoding active BMP-13 proteins will have a nucleotide sequencecomprising the nucleotide sequence beginning at any of nucleotides #410,458, 602, 605 or 659, to nucleotide #961 or 964 of SEQ ID NO:25.

In order to produce the purified tendon/ligament-like tissue inducingproteins useful for the present invention, a method is employedcomprising culturing a host cell transformed with a DNA sequencecomprising a suitable coding sequence, particularly the DNA codingsequence from nucleotide #496, #571 or # 577 to #879 or #882 of SEQ IDNO:1; and recovering and purifying from the culture medium a proteinwhich contains the amino acid sequence or a substantially homologoussequence as represented by amino acids #−25, #1 or #3 to #103 or #104 ofSEQ ID NO:2. In another embodiment, the method employed comprisesculturing a host cell transformed with a DNA sequence comprising asuitable coding sequence, particularly the DNA coding sequence fromnucleotide #605 or # 659 to #961 or #964 of SEQ ID NO:25; and recoveringand purifying from the culture medium a protein which contains the aminoacid sequence or a substantially homologous sequence as represented byamino acids #1 or #19 to #119 or #120 of SEQ ID NO:26.

The human MP52 DNA is described in W093/16099, the disclosure of whichis incorporated herein by reference. However, this document does notdisclose the ability of the protein to form tendon/ligament-like tissue,or its use in compositions for induction of tendon/ligament-like tissue.Human MP52 was originally isolated using RNA from human embryo tissue.The human MP52 nucleotide sequence (SEQ ID NO:3) and the encoded aminoacid sequences (SEQ ID NO:4) are set forth in the Sequence Listingsherein. The MP52 protein appears to begin at nucleotide #845 of SEQ IDNO:3 and continues through nucleotide #1204 of SEQ ID NO:3. The firstcysteine of the seven cysteine structure characteristic of TGF-βproteins begins at nucleotide #899. The last cysteine ends at #1201.Other active species of MP52 protein may have additional nucleotides atthe N-terminal direction from nucleotide #845 of SEQ ID NO:3.

Purified human MP52 proteins of the present invention may be produced byculturing a host cell transformed with a DNA sequence comprising the DNAcoding sequence of SEQ ID NO:3 from nucleotide #845 to #1204, andrecovering and purifying from the culture medium a protein whichcontains the amino acid sequence or a substantially homologous sequenceas represented by amino acids #1 to #120 of SEQ ID NO:4. It is alsoexpected that the amino acid sequence from amino acids #17 or #19 to#119 or #120 of SEQ ID NO:4 will retain activity. Thus, the DNA sequencefrom nucleotides #845, #893 or #899 to #1201 or #1204 are expected toencode active proteins.

For expression of the protein in mammalian host cells, the host cell istransformed with a coding sequence encoding a propeptide suitable forthe secretion of proteins by the host cell is linked in proper readingframe to the coding sequence for the mature protein. For example, seeU.S. Pat. No.5,168,050, the disclosure of which is hereby incorporatedby reference, in which a DNA encoding a precursor portion of a mammalianprotein other than BMP-2 is fused to the DNA encoding a mature BMP-2protein. Thus, the present invention includes chimeric DNA moleculescomprising a DNA sequence encoding a propeptide from a member of theTGF-β superfamily of proteins, is linked in correct reading frame to aDNA sequence encoding a tendon/ligament-like tissue inducingpolypeptide. The term “chimeric” is used to signify that the propeptideoriginates from a different polypeptide than the encoded maturepolypeptide. Of course, the host cell may be transformed with a DNAsequence coding sequence encoding the native propeptide linked incorrect reading frame to a coding sequence encoding the mature proteinshown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:26. The full sequence ofthe native propeptide may be determined through methods known in the artusing the sequences disclosed in SEQ ID NO:1, SEQ ID NO:3, or SEQ IDNO:25 to design a suitable probe for identifying and isolating theentire clone.

The present invention also encompasses the novel DNA sequences, free ofassociation with DNA sequences encoding other proteinaceous materials,and coding for expression of tendon/ligament-like tissue inducingproteins. These DNA sequences include those depicted in SEQ ID NO:1 in a5′ to 3′ direction and those sequences which hybridize thereto understringent hybridization conditions [for example, 0.1×SSC, 0.1% SDS at65° C.; see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual),Cold Spring Harbor Laboratory (1982), pages 387 to 389] and encode aprotein having tendon/ligament-like tissue inducing activity.

Similarly, DNA sequences which code for proteins coded for by thesequences of SEQ ID NO:1 or SEQ ID NO:25, or proteins which comprise theamino acid sequence of SEQ ID NO:2 or SEQ ID NO:26, but which differ incodon sequence due to the degeneracies of the genetic code or allelicvariations (naturally-occurring base changes in the species populationwhich may or may not result in an amino acid change) also encode thetendon/ligament-like tissue inducing proteins described herein.Variations in the DNA sequences of SEQ ID NO:1 or SEQ ID NO:25 which arecaused by point mutations or by induced modifications (includinginsertion, deletion, and substitution) to enhance the activity,half-life or production of the polypeptides encoded are also encompassedin the invention.

Another aspect of the present invention provides a novel method forproducing tendon/ligament-like tissue inducing proteins. The method ofthe present invention involves culturing a suitable cell line, which hasbeen transformed with a DNA sequence encoding a protein of theinvention, under the control of known regulatory sequences. Thetransformed host cells are cultured and the proteins recovered andpurified from the culture medium. The purified proteins aresubstantially free from other proteins with which they are co-producedas well as from other contaminants.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO). As described above, expression of protein inmammalian cells requires an appropriate propeptide to assure secretionof the protein. The selection of suitable mammalian host cells andmethods for transformation, culture, amplification, screening, productproduction and purification are known in the art. See, e.g., Gething andSambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman et al,Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al, U.S. Pat. No.4,419,446. Another suitable mammalian cell line, which is described inthe accompanying examples, is the monkey COS-1 cell line. The mammaliancell CV-1 may also be suitable.

Bacterial cells may also be suitable hosts. For example, the variousstrains of E. coli (e.g., HB101, MC1061) are well-known as host cells inthe field of biotechnology. Various strains of B. subtilis, Pseudomonas,other bacilli and the like may also be employed in this method. Forexpression of the protein in bacterial cells, DNA encoding a propeptideis not necessary.

Bacterial expression of mammalian proteins, including members of theTGF-β family is known to produce the proteins in a non-glycosylatedform, and in the form of insoluble pellets, known as inclusion bodies.Techniques have been described in the art for solubilizing theseinclusion bodies, denaturing the protein using a chaotropic agent, andrefolding the protein sufficiently correctly to allow for theirproduction in a soluble form. For example, see EP 0433225, thedisclosure of which is hereby incorporated by reference.

Alternatively, methods have been devised which circumvent inclusion bodyformation, such as expression of gene fusion proteins, wherein thedesired protein is expressed as a fusion protein with a fusion partner.The fusion protein is later subjected to cleavage to produce the desiredprotein. One example of such a gene fusion expression system for E. coliis based on use of the E. coli thioredoxin gene as a fusion partner,LaVallie et al., Bio/Technology, 11:187-193 (1993), the disclosure ofwhich is hereby incorporated by reference.

Many strains of yeast cells known to those skilled in the art may alsobe available as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

Another aspect of the present invention provides vectors for use in themethod of expression of these tendon/ligament-like tissue inducingproteins. Preferably the vectors contain the full novel DNA sequencesdescribed above which encode the novel factors of the invention.Additionally, the vectors contain appropriate expression controlsequences permitting expression of the protein sequences. Alternatively,vectors incorporating modified sequences as described above are alsoembodiments of the present invention. Additionally, the sequence of SEQID NO:1 or SEQ ID NO:3 or SEQ ID NO:25 could be manipulated to express amature protein by deleting propeptide sequences and replacing them withsequences encoding the complete propeptides of BMP proteins or membersof the TGF-β superfamily. Thus, the present invention includes chimericDNA molecules encoding a propeptide from a member of the TGF-βsuperfamily linked in correct reading frame to a DNA sequence encoding aprotein having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 orSEQ ID NO:26, The vectors may be employed in the method of transformingcell lines and contain selected regulatory sequences in operativeassociation with the DNA coding sequences of the invention which arecapable of directing the replication and expression thereof in selectedhost cells. Regulatory sequences for such vectors are known to thoseskilled in the art and may be selected depending upon the host cells.Such selection is routine and does not form part of the presentinvention.

A protein of the present invention, which induces tendon/ligament-liketissue or other tissue formation in circumstances where such tissue isnot normally formed, has application in the healing of tendon orligament tears, deformities and other tendon or ligament defects inhumans and other animals. Such a preparation employing atendon/ligament-like tissue inducing protein may have prophylactic usein preventing damage to tendon or ligament tissue, as well as use in theimproved fixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of thepresent invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the invention may also beuseful in the treatment of tendinitis, carpal tunnel syndrome and othertendon or ligament defects. The compositions of the present inventioncan also be used in other indications wherein it is desirable to heal orregenerate tendon and/or ligament tissue. Such indications include,without limitation, regeneration or repair of injuries to theperiodontal ligament, such as occurs in tendonitis, and regeneration orrepair of the tendon-to-bone attachment. The compositions of the presentinvention may provide an environment to attract tendon- orligament-forming cells, stimulate growth of tendon- or ligament-formingcells or induce differentiation of progenitors of tendon- orligament-forming cells.

The BMP-12 related proteins may be recovered from the culture medium andpurified by isolating them from other proteinaceous materials from whichthey are co-produced and from other contaminants present. The proteinsof the present invention are capable of inducing the formation oftendon/ligament-like tissue. These proteins may be further characterizedby the ability to demonstrate tendon/ligament-like tissue formationactivity in the rat ectopic implant assay described below. It iscontemplated that these proteins may have ability to induce theformation of other types of tissue, such as ligaments, as well.

The tendon/ligament-like tissue inducing proteins provided herein alsoinclude factors encoded by the sequences similar to those of SEQ ID NO:1or SEQ ID NO:25, but into which modifications are naturally provided(e.g. allelic variations in the nucleotide sequence which may result inamino acid changes in the polypeptide) or deliberately engineered. Forexample, synthetic polypeptides may wholly or partially duplicatecontinuous sequences of the amino acid residues of SEQ ID NO:2. Thesesequences, by virtue of sharing primary, secondary, or tertiarystructural and conformational characteristics with tendon/ligament-liketissue growth factor polypeptides of SEQ ID NO:2 may possesstendon/ligament-like or other tissue growth factor biological propertiesin common therewith. Thus, they may be employed as biologically activesubstitutes for naturally-occurring tendon/ligament-like tissue inducingpolypeptides in therapeutic compositions and processes.

Other specific mutations of the sequences of tendon/ligament-like tissueinducing proteins described herein involve modifications ofglycosylation sites. These modifications may involve O-linked orN-linked glycosylation sites. For instance, the absence of glycosylationor only partial glycosylation results from amino acid substitution ordeletion at asparagine-linked glycosylation recognition sites. Theasparagine-linked glycosylation recognition sites comprise tripeptidesequences which are specifically recognized by appropriate cellularglycosylation enzymes. These tripeptide sequences may beasparagine-X-threonine, asparagine-X-serine or asparagine-X-cysteine,where X is usually any amino acid except proline. A variety of aminoacid substitutions or deletions at one or both of the first or thirdamino acid positions of a glycosylation recognition site (and/or aminoacid deletion at the second position) results in non-glycosylation atthe modified tripeptide sequence. Additionally, bacterial expression ofprotein will also result in production of a non-glycosylated protein,even if the glycosylation sites are left unmodified.

The compositions of the present invention comprise a purified BMP-12related protein which may be produced by culturing a cell transformedwith the DNA sequence of SEQ ID NO:1 or SEQ ID NO:25 and recovering andpurifying protein having the amino acid sequence of SEQ ID NO:2 or SEQID NO:26 from the culture medium. The purified expressed protein issubstantially free from other proteinaceous materials with which it isco-produced, as well as from other contaminants. The recovered purifiedprotein is contemplated to exhibit tendon/ligament-like tissue formationactivity, and other tissue growth activity, such as ligamentregeneration. The proteins of the invention may be further characterizedby the ability to demonstrate tendon/ligament-like tissue formationactivity in the rat assay described below.

The compositions for inducing tendon/ligament-like tissue formation ofthe present invention may comprise an effective amount of atendon/ligament-like tissue inducing protein, wherein said proteincomprises the amino acid sequence of SEQ ID NO:2, preferably amino acids#−25, #1 or #3 to #103 or #104 of SEQ ID NO:2; or amino acids #1 or #19to #120 of SEQ ID NO:26; as well as mutants and/or variants of SEQ IDNO:2 or SEQ ID NO:26, which exhibit the ability to form tendon and/orligament like tissue.

Compositions of the present invention may further comprise additionalproteins, such as additional members of the TGF-β superfamily ofproteins, such as activins. Another aspect of the invention providespharmaceutical compositions containing a therapeutically effectiveamount of a tendon/ligament-inducing protein, such as BMP-12 or VL-1, ina pharmaceutically acceptable vehicle or carrier. These compositions maybe used to induce the formation of tendon/ligament-like tissue or othertissue. It is contemplated that such compositions may also be used fortendon and ligament repair, wound healing and other tissue repair, suchas skin repair. It is further contemplated that proteins of theinvention may increase neuronal survival and therefore be useful intransplantation and treatment of conditions exhibiting a decrease inneuronal survival. Compositions of the invention may further include atleast one other therapeutically useful agent, such as the BMP proteinsBMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, disclosed forinstance in U.S. Pat. Nos. 5,108,922; 5,013,649; 5,116,738; 5,106,748;5,187,076, and 5,141,905; BMP-8, disclosed in PCT publicationW091/18098; BMP-9, disclosed in PCT publication W093/00432; and BMP-10or BMP-11, disclosed in co-pending patent applications, serial number08/061,695 presently abandoned, a continuation-in-part of which hasissued as U.S. Pat. No. 5,637,480, and 08/061,464 presently abandoned, acontinuation-in-part of which has issued as U.S. Pat. No. 5,639,638filed on May 12, 1993. The disclosure of the above documents are herebyincorporated by reference herein.

The compositions of the invention may comprise, in addition to atendon/ligament-inducing protein such as BMP-12 or VL-1 (BMP-13), othertherapeutically useful agents including MP52, epidermal growth factor(EGF), fibroblast growth factor (FGF), platelet derived growth factor(PDGF), transforming growth factors (TGF-α and TGF-β), and fibroblastgrowth factor-4 (FGF-4), parathyroid hormone (PTH), leukemia inhibitoryfactor (LIF/HILDA/DIA), insulin-like growth factors (IGF-I and IGF-II).Portions of these agents may also be used in compositions of the presentinvention. For example, a composition comprising both BMP-2 and BMP-12implanted together gives rise to both bone and tendon/ligament-liketissue. Such a composition may be useful for treating defects of theembryonic joint where tendon, ligaments, and bone form simultaneously atcontiguous anatomical locations, and may be useful for regeneratingtissue at the site of tendon attachment to bone. It is contemplated thatthe compositions of the invention may also be used in wound healing,such as skin healing and related tissue repair. The types of woundsinclude, but are not limited to burns, incisions and ulcers. (See, e.g.PCT Publication W084/01106 for discussion of wound healing and relatedtissue repair).

It is expected that the proteins of the invention may act in concertwith or perhaps synergistically with other related proteins and growthfactors. Further therapeutic methods and compositions of the inventiontherefore comprise a therapeutic amount of at least one protein of theinvention with a therapeutic amount of at least one of the BMP proteinsdescribed above. Such compositions may comprise separate molecules ofthe BMP proteins or heteromolecules comprised of different BMP moieties.For example, a method and composition of the invention may comprise adisulfide linked dimer comprising a BMP-12 related protein subunit and asubunit from one of the “BMP” proteins described above. Thus, thepresent invention includes compositions comprising a purified BMP-12related polypeptide which is a heterodimer wherein one subunit comprisesthe amino acid sequence from amino acid #1 to amino acid #104 of SEQ IDNO:2, and one subunit comprises an amino acid sequence for a bonemorphogenetic protein selected from the group consisting of BMP-1,BMP-2, BMP-3, BMP4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10 andBMP-11. A further embodiment may comprise a heterodimer of disulfidebonded tendon/ligament-like tissue inducing moieties such as BMP-12,VL-1 (BMP-13) or MP52. For example the heterodimer may comprise onesubunit comprising an amino acid sequence from #1 to # 104 of SEQ IDNO:2 and the other subunit may comprise an amino acid sequence from #1to #120 of SEQ ID NO:4 or #1 to #120 of SEQ ID NO:26. Further,compositions of the present invention may be combined with other agentsbeneficial to the treatment of the defect, wound, or tissue in question.

The preparation and formulation of such physiologically acceptableprotein compositions, having due regard to pH, isotonicity, stabilityand the like, is within the skill of the art. The therapeuticcompositions are also presently valuable for veterinary applications dueto the lack of species specificity in TGF-β proteins. Particularlydomestic animals and thoroughbred horses in addition to humans aredesired patients for such treatment with the compositions of the presentinvention.

The therapeutic method includes administering the composition topically,systemically, or locally as an injectable and/or implant or device. Whenadministered, the therapeutic composition for use in this invention is,of course, in a pyrogen-free, physiologically acceptable form. Further,the composition, may desirably be encapsulated or injected in a viscousform for delivery to the site of tissue damage. Topical administrationmay be suitable for wound healing and tissue repair. Therapeuticallyuseful agents other than the proteins which may also optionally beincluded in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with thecomposition in the methods of the invention. In addition, thecompositions of the present invention may be used in conjunction withpresently available treatments for tendon/ligament injuries, such assuture (e.g., vicryl sutures or surgical gut sutures, Ethicon Inc.,Somerville, N.J.) or tendon/ligament allograft or autograft, in order toenhance or accelerate the healing potential of the suture or graft. Forexample, the suture, allograft or autograft may be soaked in thecompositions of the present invention prior to implantation. It may alsobe possible to incorporate the protein or composition of the inventiononto suture materials, for example, by freeze-drying.

The compositions may include an appropriate matrix and/or sequesteringagent as a carrier. For instance, the matrix may support the compositionor provide a surface for tendon/ligament-like tissue formation and/orother tissue formation. The matrix may provide slow release of theprotein and/or the appropriate environment for presentation thereof. Thesequestering agent may be a substance which aids in ease ofadministration through injection or other means, or may slow themigration of protein from the site of application.

The choice of a carrier material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are nonbiodegradable and chemicallydefined. Preferred matrices include collagen-based materials, includingsponges, such as Helistat® (Integra LifeSciences, Plainsboro, N.J.), orcollagen in an injectable form, as well as sequestering agents, whichmay be biodegradable, for example hyalouronic acid derived.Biodegradable materials, such as cellulose films, or surgical meshes,may also serve as matrices. Such materials could be sutured into aninjury site, or wrapped around the tendon/ligament.

Another preferred class of carrier are polymeric matrices, includingpolymers of poly(lactic acid), poly(glycolic acid) and copolymers oflactic acid and glycolic acid. These matrices may be in the form of asponge, or in the form of porous particles, and may also include asequestering agent. Suitable polymer matrices are described, forexample, in W093/00050, the disclosure of which is incorporated hereinby reference.

Preferred families of sequestering agents include blood, fibrin clotand/or cellulosic materials such as alkylcelluloses (includinghydroxyalkylcelluloses), including methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropyl-methylcellulose, and carboxymethylcellulose, the mostpreferred being cationic salts of carboxymethylcellulose (CMC). Otherpreferred sequestering agents include hyaluronic acid, sodium alginate,poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer andpoly(vinyl alcohol). The amount of sequestering agent useful herein is0.5-20 wt %, preferably 1-10 wt % based on total formulation weight,which represents the amount necessary to prevent desorbtion of theprotein from the polymer matrix and to provide appropriate handling ofthe composition, yet not so much that the progenitor cells are preventedfrom infiltrating the matrix, thereby providing the protein theopportunity to assist the activity of the progenitor cells.

Additional optional components useful in the practice of the subjectapplication include, e.g. cryogenic protectors such as mannitol,sucrose, lactose, glucose, or glycine (to protect the protein fromdegradation during lyophilization), antimicrobial preservatives such asmethyl and propyl parabens and benzyl alcohol; antioxidants such asEDTA, citrate and BHT (butylated hydroxytoluene); and surfactants sifdchas poly(sorbates) and poly(oxyethylenes); etc.

As described above, the compositions of the invention may be employed inmethods for treating a number of tendon defects, such as theregeneration of tendon/ligament-like tissue in areas of tendon orligament damage, to assist in repair of tears of tendon tissue,ligaments, and various other types of tissue defects or wounds. Thesemethods, according to the invention, entail administering to a patientneeding such tendon/ligament-like tissue or other tissue repair, acomposition comprising an effective amount of a tendon/ligament-liketissue inducing protein; such as described in SEQ ID NO:2, SEQ ID NO:4and/or SEQ ID NO:26. These methods may also entail the administration ofa tendon/ligament-like tissue inducing protein in conjunction with atleast one of the BMP proteins described above.

In another embodiment, the methods may entail administration of aheterodimeric protein in which one of the monomers is atendon/ligament-like tissue inducing polypeptide, such as BMP-12, VL-1(BMP-13) or MP52, and the second monomer is a member of the TGF-βsuperfamily of growth factors. In addition, these methods may alsoinclude the administration of a tendon/ligament-like tissue inducingprotein with other growth factors including EGF, FGF, TGF-α, TGF-β, andIGF.

Thus, a further aspect of the invention is a therapeutic method andcomposition for repairing tendon/ligament-like tissue, for repairingtendon or ligament as well as treating tendinitis and other conditionsrelated to tendon or ligament defects. Such compositions comprise atherapeutically effective amount of one or more tendon/ligament-liketissue inducing proteins, such as BMP-12, a BMP-12 related protein, orMP52, in admixture with a pharmaceutically acceptable vehicle, carrieror-matrix.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the composition,e.g., amount of tendon or ligament tissue desired to be formed, the siteof tendon or ligament damage, the condition of the damaged tendon orligament, the size of a wound, type of damaged tissue, the patient'sage, sex, and diet, the severity of any infection, time ofadministration and other clinical factors. The dosage may vary with thetype of matrix used in the reconstitution and the types of additionalproteins in the composition. The addition of other known growth factors,such as IGF-I (insulin like growth factor I), to the final composition,may also affect the dosage.

Progress can be monitored by periodic assessment of tendon/ligament-liketissue formation, or tendon or ligament growth and/or repair. Theprogress can be monitored by methods known in the art, for example,X-rays, arthroscopy, histomorphometric determinations and tetracyclinelabeling.

The following examples illustrate practice of the present invention inrecovering and characterizing human tendon/ligament-like tissue inducingprotein and employing them to recover the other tendon/ligament-liketissue inducing proteins, obtaining the human proteins, expressing theproteins via recombinant techniques, and demonstration of the ability ofthe compositions of the present invention to form tendon/ligament-liketissue in an in vivo model. Although the examples demonstrate theinvention with respect to BMP-12, with minor modifications within theskill of the art, the same results are believed to be attainable withMP52 and VL-1.

EXAMPLE 1 Isolation of DNA

DNA sequences encoding BMP-12 and BMP-12 related proteins may beisolated by various techniques known to those skilled in the art. Asdescribed below, oligonucleotide primers may be designed on the basis ofamino acid sequences present in other BMP proteins, Vg-1 relatedproteins and other proteins of the TGF-β superfamily. Regions containingamino acid sequences which are highly conserved within the BMP family ofproteins and within other members of the TGF-β superfamily of proteinscan be identified and consensus amino acid sequences of these highlyconserved regions can be constructed based on the similarity of thecorresponding regions of individual BMP/TGF-β/Vg-1 proteins. An exampleof such a consensus amino acid sequence is indicated below. Consensusamino acid sequence (1):

Trp-Gln/Asn-Asp-Trp-Ile-Val/Ile-Ala (SEQ ID NO:16) Where X/Y indicatesthat either amino acid residue may appear at that position.

The following oligonucleotide is designed on the basis of the aboveidentified consensus amino acid sequence (1):

#1: CGGATCCTGGVANGAYTGGATHRTNGC (SEQ ID NO:17)

This oligonucleotide sequence is synthesized on an automated DNAsynthesizer. The standard nucleotide symbols in the above identifiedoligonucleotide primer are as follows: A,adenosine; C,cytosine;G,guanine; T,thymine; N,adenosine or cytosine or guanine or thymine;R,adenosine or cytosine; Y,cytosine or thymine; H,adenosine or cytosineor thymine; V,adenosine or cytosine or guanine; D,adenosine or guanineor thymine.

The first seven nucleotides of oligonucleotide #1 (underlined) containthe recognition sequence for the restriction endonuclease BamHI in orderto facilitate the manipulation of a specifically amplified DNA sequenceencoding the BMP-12 protein and are thus not derived from the consensusamino acid sequence (1) presented above.

A second consensus amino acid sequence is derived from another highlyconserved region of BMP/TGF-β/Vg-1 proteins as described below:

His-Ala-Ile-Val/Leu-Gln-Thr (SEQ ID NO:18)

The following oligonucleotide is designed on the basis of the aboveidentified consensus amino acid sequence (2):

#2: TTTCTAGAARNGTYTGNACDATNGCRTG (SEQ ID NO:19)

This oligonucleotide sequence is synthesized on an automated DNAsynthesizer. The same nucleotide symbols are used as described above.

The first seven nucleotides of oligonucleotide #1 (underlined) containthe recognition sequence for the restriction endonuclease XbaI in orderto facilitate the manipulation of a specifically amplified DNA sequenceencoding the BMP-12 protein and are thus not derived from the consensusamino acid sequence (2) presented above.

It is contemplated that the BMP-12 protein of the invention and otherBMP/TGF-β/Vg-1 related proteins may contain amino acid sequences similarto the consensus amino acid sequences described above and that thelocation of those sequences within a BMP-12 protein or other novelrelated proteins would correspond to the relative locations in theproteins from which they were derived. It is further contemplated thatthis positional information derived from the structure of otherBMP/TGF-β/Vg-1 proteins and the oligonucleotide sequences #1 and #2which have been derived from consensus amino acid sequences (1) and (2),respectively, could be utilized to specifically amplify DNA sequencesencoding the corresponding amino acids of a BMP-12 protein or otherBMP/TGF-β/Vg-1 related proteins.

Based on the knowledge of the gene structures of BMP/TGF-β/Vg- 1proteins it is further contemplated that human genomic DNA can be usedas a template to perform specific amplification reactions which wouldresult in the identification of BMP-12 BMP/TGF-β/Vg-1 (BMP-12 relatedprotein) encoding sequences. Such specific amplification reactions of ahuman genomic DNA template could be initiated with the use ofoligonucleotide primers #1 and #2 described earlier. Oligonucleotides #1and #2 identified above are utilized as primers to allow the specificamplification of a specific nucleotide sequence from human genomic DNA.The amplification reaction is performed as follows:

Human genomic DNA (source: peripheral blood lymphocytes), provided byKen Jacobs of Genetics Institute, is sheared by repeated passage througha 25 gauge needle, denatured at 100° C. for 5 minutes and then chilledon ice before adding to a reaction mixture containing 200 μM eachdeoxynucleotide triphosphates (dATP, dGTP, dCTP and dTTP), 10 mMTris-HCl pH 8.3, 50 mM KCI, 1.5 mM MgCl₂, 0.001% gelatin, 1.25 units TaqDNA polymerase, 100 pM oligonucleotide #1 and 100 pM oligonucleotide #2.This reaction mixture is incubated at 94° C. for two minutes and thensubjected to thermal cycling in the following manner: 1 minute at 94°C., 1 minute at 40° C., 1 minute at 72° C. for three cycles; then 1minute at 94° C. 1 minute at 55° C., 1 minute at 72° C. for thirty-sevencycles, followed by a 10 minute incubation at 72° C.

The DNA which is specifically amplified by this reaction is ethanolprecipitated, digested with the restriction endonucleases BamHI and XbaIand subjected to agarose gel electrophoresis. A region of the gel,corresponding to the predicted size of the BMP-12 or otherBMP/TGF-β/Vg-1 encoding DNA fragment, is excised and the specificallyamplified DNA fragments contained therein are electroeluted andsubcloned into the plasmid vector pGEM-3 between the XbaI and BamHIsites of the polylinker. DNA sequence analysis of one of the resultingBMP-12 related subclones indicates the specifically amplified DNAsequence product contained therein encodes a portion of the BMP-12protein of the invention.

The DNA sequence (SEQ ID NO:5) and derived amino acid sequence (SEQ IDNO:6) of this specifically amplified DNA fragment of BMP-12 are shown inthe SEQUENCE Listings.

Nucleotides #1-#26 of SEQ ID NO:5 comprise a portion of oligonucleotide#1 and nucleotides #103-#128 comprise a portion of the reversecompliment of oligonucleotide #2 utilized to perform the specificamplification reaction. Due to the function of oligonucleotides #1 and#2 in initiating the amplification reaction, they may not correspondexactly to the actual sequence encoding a BMP-12 protein and aretherefore not translated in the corresponding amino acid derivation (SEQID NO:6).

DNA sequence analysis of another subclone indicates that thespecifically amplified DNA product contained therein encodes a portionof another BMP/TGF-β/Vg-1 (BMP-12 related) protein of the inventionnamed VL-1.

The DNA sequence (SEQ ID NO:7) and derived amino acid sequence (SEQ IDNO:8) of this specifically amplified DNA fragment are shown in theSequence Listings.

Nucleotides #1-#26 of SEQ ID NO:7 comprise a portion of oligonucleotide#1 and nucleotides #103-#128 comprise a portion of the reversecompliment of oligonucleotide #2 utilized to perform the specificamplification reaction. Due to the function of oligonucleotides #1 and#2 in initiating the amplification reaction, they may not correspondexactly to the actual sequence encoding a VL-1 protein of the inventionand are therefore not translated in the corresponding amino acidderivation (SEQ ID NO:8).

The following oligonucleotide probe is designed on the basis of thespecifically amplified BMP-12 human DNA sequence set forth above (SEQ IDNO:5) and synthesized on an automated DNA synthesizer:

#3: CCACTGCGAGGGCCTTTGCGACTTCCCTTTGCGTTCGCAC (SEQ ID NO:20)

This oligonucleotide probe is radioactively labeled with ³²P andemployed to screen a human genomic library constructed in the vectorλFIX (Stratagene catalog #944201). 500,000 recombinants of the humangenomic library are plated at a density of approximately 10,000recombinants per plate on 50 plates. Duplicate nitrocellulose replicasof the recombinant bacteriophage plaques and hybridized tooligonucleotide probe #3 in standard hybridization buffer (SHB=5×SSC,0.1% SDS, 5×Denhardt's, 100 μg/ml salmon sperm DNA) at 65° C. overnight.The following day the radioactively labelled oligonucleotide containinghybridization solution is removed an the filters are washed with0.2×SSC, 0.1% SDS at 65° C. A single positively hybridizing recombinantis identified and plaque purified. This plaque purified recombinantbacteriophage clone which hybridizes to the BMP-12 oligonucleotide probe#3 is designated λHuG-48. A bacteriophage plate stock is made andbacteriophage DNA is isolated from the λHuG-48 human genomic clone. Thebacteriophage λHuG-48 has been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. “ATCC” under theaccession #75625 on Dec. 7, 1993. This deposit meets the requirements ofthe Budapest Treaty of the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure and Regulationsthereunder. The oligonucleotide hybridizing region of this recombinant,λHuG-48, is localized to a 3.2 kb BamHI fragment. This fragment issubcloned into a plasmid vector (pGEM-3) and DNA sequence analysis isperformed. This plasmid subdlone is designated PCR1-1#2 and has beendeposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. “ATCC” under the accession #69517 on Dec. 7, 1993.This deposit meets the requirements of the Budapest Treaty of theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and Regulations thereunder. The partial DNAsequence (SEQ ID NO:1) and derived amino acid sequence (SEQ ID NO:2) ofthe 3.2 kb DNA insert of the plasmid subclone PCR1-1#2, derived fromclone λHuG-48, are shown in the Sequence Listings.

It should be noted that nucleotides #639-#714 of SEQ ID NO:1 correspondto nucleotides #27-#102 of the specifically amplified BMP-12 encodingDNA fragment set forth in SEQ ID NO:5 thus confirming that the humangenomic bacteriophage clone λHuG-48 and derivative subclone PCR1-1#2encode at least a portion of the BMP-12 protein of the invention. Thenucleotide sequence of a portion of the 3.2 kb BamHI insert of theplasmid PCR1-1#2 contains an open reading frame of at least 882 basepairs, as defined by nucleotides #1-#882 of SEQ ID NO:1. This openreading frame encodes at least 294 amino acids of the human BMP-12protein of the invention. The encoded 294 amino acid human BMP-12protein includes the full mature human BMP-12 protein (amino acids#1-#104 of SEQ ID NO:2), as well as the C-terminal portion of thepropeptide region of the primary translation product (amino acid #−190to #−1 of SEQ ID NO:2).

Additional DNA sequence of the 3.2 kb BamHI insert of the plasmidPCR1-1#2 set forth in SEQ ID NO:33 demonstrates the presence of an 1164bp open reading frame, as defined by nucleotides #138 through #1301 ofSEQ ID NO:33. [NOTE that all the sequence disclosed in SEQ ID NO:1 iscontained within SEQ ID NO:33]. As this sequence is derived from agenomic clone it is difficult to determine the boundary between the 5′extent of coding sequence and the 3′ limit of intervening sequence(intron/non-coding sequence).

Based on the knowledge of other BMP proteins and other proteins withinthe TGF-β family, it is predicted that the precursor polypeptide wouldbe cleaved at the multibasic sequence Arg-Arg-Gly-Arg (SEQ ID NO:2,residues −4 to −1) in agreement with a proposed consensus proteolyticprocessing sequence of Arg-X-X-Arg (SEQ ID NO:2, residues −4 to −1).Cleavage of the BMP-12 precursor polypeptide is expected to generate a104 amino acid mature peptide beginning with the amino acid Ser atposition #1 of SEQ ID NO:2. The processing of BMP-12 into the matureform is expected to involve dimerization and removal of the N-terminalregion in a manner analogous to the processing of the related proteinTGF-β [Gentry et al., Molec & Cell. Biol., 8:4162 (1988); Derynck et al.Nature, 316:701 (1985)].

It is contemplated therefore that the mature active species of BMP-12comprises a homodimer of two polypeptide subunits, each subunitcomprising amino acids #1 to #104 of SEQ ID NO:2 with a predictedmolecular weight of approximately 12,000 daltons. Further active speciesare contemplated comprising at least amino acids #3 to #103 of SEQ IDNO:2, thereby including the first and last conserved cysteine residue.As with other members of the TGF-β/BMP family of proteins, thecarboxy-terminal portion of the BMP-12 protein exhibits greater sequenceconservation than the more amino-terminal portion. The percent aminoacid identity of the human BMP-12 protein in the cysteine-richC-terminal domain (amino acids #3-#104) to the corresponding region ofhuman BMP proteins and other proteins within the TGF-β family is asfollows: BMP-2, 55%; BMP-3, 43%; BMP-4, 53%; BMP-5, 49%; BMP-6, 49%;BMP-7, 50%; BMP-8, 57%; BMP-9, 48%; BMP-10, 57%; activin WC (BMP-11),38%; Vg1, 46%; GDF-1, 47%; TGF-β1, 36%; TGF-β2, 36%; TGF-β3, 39%;inhibin β(B), 36%; inhibin, β(A), 41%.

The human BMP-12 DNA sequence (SEQ ID NO:1), or a portion thereof, canbe used as a probe to identify a human cell line or tissue whichsynthesizes BMP-12 mRNA. Briefly described, RNA is extracted from aselected cell or tissue source and either electrophoresed on aformaldehyde agarose gel and transferred to nitrocellulose, or reactedwith formaldehyde and spotted on nitrocellulose directly. Thenitrocellulose is then hybridized to a probe derived from the codingsequence of human BMP-12.

Alternatively, the human BMP-12 sequence is used to designoligonucleotide primers which will specifically amplify a portion of theBMP-12 encoding sequence located in the region between the primersutilized to perform the specific amplification reaction. It iscontemplated that these human BMP-12 derived primers would allow one tospecifically amplify corresponding BMP-12 encoding sequences from MRNA,cDNA or genomic DNA templates. Once a positive source has beenidentified by one of the above described methods, mRNA is selected byoligo (dT) cellulose chromatography and cDNA is synthesized and clonedin λgt10 or other λ bacteriophage vectors known to those skilled in theart, for example, λZAP by established techniques (Toole et al., supra).It is also possible to perform the oligonucleotide primer directedamplification reaction, described above, directly on a pre-establishedhuman cDNA or genomic library which has been cloned into a λbacteriophage vector. In such cases, a library which yields aspecifically amplified DNA product encoding a portion of the humanBMP-12 protein could be screened directly, utilizing the fragment ofamplified BMP-12 encoding DNA as a probe.

Oligonucleotide primers designed on the basis of the DNA sequence of thehuman BMP-12 genomic clone λHuG-48 are predicted to allow the specificamplification of human BMP-12 encoding DNA sequences frompre-established human cDNA libraries which are commercially available(ie. Stratagene, La Jolla, Calif. or Clontech Laboratories, Inc., PaloAlto, Calif.). The following oligonucleotide primer is designed on thebasis of nucleotides #571 to #590 of the DNA sequence set forth in SEQID NO:1 and synthesized on an automated DNA synthesizer:

#4: TGCGGATCCAGCCGCTGCAGCCGCAAGCC (SEQ ID NO:21)

The first nine nucleotides of primer #4 (underlined) comprise therecognition sequence for the restriction endonuclease BamHI which can beused to facilitate the manipulation of a specifically amplified DNAsequence encoding the human BMP-12 protein of the invention and are thusnot derived from the DNA sequence presented in SEQ ID NO:1. Thefollowing oligonucleotide primer is designed on the basis of nucleotides#866-#885 of the DNA sequence set forth in SEQ ID NO:1 and synthesizedon an automated DNA synthesizer:

#5GACTCTAGACTACCTGCAGCCGCAGGCCT (SEQ ID NO:22)

The first nine nucleotides of primer #5 (underlined) comprise therecognition sequence for the restriction endonuclease XbaI which can beused to facilitate the manipulation of a specifically amplified DNAsequence encoding the human BMP-12 protein of the invention and are thusnot derived from the DNA sequence presented in SEQ ID NO:1.

The standard nucleotide symbols in the above identified primers are asfollows: A, adenine; C, cytosine; G, guanine; T, thymine.

Primers #4 and #5 identified above are utilized as primers to allow theamplification of a specific BMP-12 encoding nucleotide sequence frompre-established cDNA libraries which may include the following: humanfetal brain cDNA/λZAPII (Stratagene catalog #936206), humanliver/λUNI-ZAP XR (Stratagene Catalog #937200), human lung/λUNI-ZAP XR(Stratagene catalog #937206), and human fetal spleen/UNI-ZAP XR(Stratagene catalog #937205).

Approximately 1×10⁸ pfu (plaque forming units) of λbacteriophagelibraries containing human cDNA inserts such as those detailed above aredenatured at 95° C. for five minutes prior to addition to a reactionmixture containing 200 μM each deoxynucleotide triphosphates (dATP,dGTP, dCTP and dTTP) 10 MM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂,0.001% gelatin, 1.25 units Taq DNA polymerase, 100 pM oligonucleotideprimer #4 and 100 pM oligonucleotide primer #5. The reaction mixture isthen subjected to thermal cycling in the following manner: 1 minute at94° C., 1 minute at 50° C., 1 minute at 72° C. for thirty-nine cyclesfollowed by 10 minutes at 72° C.

The DNA which is specifically amplified by this reaction would beexpected to generate a BMP-12 encoding product of approximately 333 basepairs, the internal 315 bp of which correspond to nucleotides #571 to#885 of SEQ ID NO:1 and also including 9 bp at each end of the BMP-12specific fragment which correspond to the restriction sites defined bynucleotides #1-#9 of primers #4 and #5. The resulting 333 bp DNA productis digested with the restriction endonucleases BamHI and XbaI, phenolextracted, chloroform extracted and ethanol precipitated.

Alternatively, to ethanol precipitation, buffer exchange and removal ofsmall fragments of DNA resulting from the BamHI/XbaI restriction digestis accomplished by dilution of the digested DNA product in 10 mMTris-HCI pH 8.0, 1 nM EDTA followed by centrifugation through aCentricon™ 30 microconcentrator (W. R. Grace & Co., Beverly, Mass.;Product #4209). The resulting BamHI/XbaI digested amplified DNA productis subcloned into a plasmid vector (ie. pBluescript, pGEM-3 etc.)between the BamHI and XbaI sites of the polylinker region. DNA sequenceanalysis of the resulting subclones would be required to confirm theintegrity of the BMP-12 encoding insert. Once a positive cDNA source hasbeen identified in this manner, the corresponding cDNA library fromwhich a 333 bp BMP-12 specific sequence was amplified could be screeneddirectly with the 333 bp insert or other BMP-12 specific probes in orderto identify and isolate cDNA clones encoding the full-length BMP-12protein of the invention.

Additional methods known to those skilled in the art may be used toisolate other full-length cDNAs encoding human BMP-12 related proteins,or full length cDNA clones encoding BMP-12 related proteins of theinvention from species other than humans, particularly other mammalianspecies.

The following examples demonstrate the use of the human BMP-12 sequenceto isolate homologues from BMP-12 related proteins in a murine genomicDNA library.

The DNA sequence which encodes the human BMP-12 protein of the inventionis predicted to be significantly homologous to BMP-12 and BMP-12 relatedsequences from species other than humans that it could be utilized tospecifically amplify DNA sequences from those other species which wouldencode the corresponding BMP-12 related proteins. Specifically, thefollowing oligonucleotides are designed on the basis of the human BMP-12sequence (SEQ ID NO:1) and are synthesized on an automated DNAsynthesizer:

#6: GCGGATCCAAGGAGCTCGGCTGGGACGA (SEQ ID NO:23)

#7: GGAATTCCCCACCACCATGTCCTCGTAT (SEQ ID NO:24)

The first eight nucleotides of oligonucleotide primers #6 and #7(underlined) comprise the recognition sequence for the restrictionendonucleases BamHI and EcoRI, respectively. These sequences areutilized to facilitate the manipulation of a specifically amplified DNAsequence encoding a BMP-12 or BMP-12 related protein from a speciesother than human and are thus not derived from the DNA sequencepresented in SEQ ID NO:1. Oligonucleotide primer #6 is designed on thebasis of nucleotides #607-#626 of SEQ ID NO:1. Oligonucleotide primer #7is designed on the basis of the reverse compliment of nucleotides#846-#865 of the DNA sequence set forth in SEQ ID NO:1.

Oligonucleotide primers #6 and #7 identified above are utilized asprimers to allow the amplification of specific BMP-12 related sequencesfrom genomic DNA derived from species other than humans. Theamplification reaction is performed as follows:

Murine genomic DNA (source: strain Balb c) is sheared by repeatedpassage through a 25 gauge needle, denatured at 100° C. for five minutesand then chilled on ice before adding to a reaction mixture containing200 μM each deoxynucleotide triphosphates (dATP, DGTP, dCTP and dTTP) 10mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, 1.25 unitsTaq DNA polymerase, 100 pM oligonucleotide primer #6 and 100 pMoligonucleotide primer #7. The reaction mixture is then subjected tothermal cycling in the following manner: 1 minute at 95° C., 1 minute at55° C., 1 minute at 72° C. for forty cycles followed by 10 minutes at72° C.

The DNA which is specifically amplified by this reaction is ethanolprecipitated, digested with the restriction endonucleases BamHI andEcoRI and subjected to agarose gel electrophoresis. A region of the gel,corresponding to the predicted size of the murine BMP-12 or BMP-12related encoding DNA fragment, is excised and the specifically amplifiedDNA fragments contained therein are extracted (by electroelution or byother methods known to those skilled in the art) and subcloned in to aplasmid vector, such as pGEM-3 or pBluescript between the BamHI andEcoRI sites of the polylinker. DNA sequence analysis of one of theresulting subclones named mVl, indicates that the specifically amplifiedDNA sequence contained therein encodes a portion of a protein whichappears to be the murine homolog to either the BMP-12 or VL-1 sequenceof the invention. The DNA sequence (SEQ ID NO:10) and derived amino acidsequence (SEQ ID NO:11) of this specifically amplified murine DNAfragment are shown in the sequence listings.

Nucleotides #1-#26 of SEQ ID NO:10 comprise a portion of oligonucleotide#6 and nucleotides #246-#272 comprise a portion of the reversecompliment of oligonucleotide #7 utilized to perform the specificamplification reaction. Nucleotide #27 of SEQ ID NO:10 appears to be thelast nucleotide of a codon triplet, and nucleotides #244-#245 of SEQ IDNO:10 appear to be the first two nucleotides of a codon triplet.Therefore, nucleotides #28 to #243 of SEQ ID NO:10 correspond to apartial coding sequence of mV1. Due to the function of oligonucleotides#6 and #7 in initiating the amplification reaction, they may notcorrespond exactly to the actual sequence encoding the murine homolog tothe human BMP-12 or VL-1 protein of the invention and are therefore nottranslated in the corresponding amino acid sequence derivation (SEQ IDNO:11).

Oligonucleotide probes designed on the basis of the specificallyamplified murine BMP-12 or VL-1 DNA sequence set forth in SEQ ID NO:10can be utilized by those skilled in the art to identify full-lengthmurine BMP-12 or VL-1 encoding clones (either cDNA or genomic).

DNA sequence analysis of another of the resulting subclones named mV2,indicates that the specifically amplified DNA sequence contained thereinencodes a portion of a murine BMP-12 related sequence of the invention.The DNA sequence (SEQ ID NO:12) and derived amino acid sequence (SEQ IDNO:13) of this specifically amplified murine DNA fragment are shown inthe sequence listings.

Nucleotides #1-#26 of SEQ ID NO:12 comprise a portion of oligonucleotide#6 and nucleotides #246-#272 comprise a portion of the reversecompliment of oligonucleotide #7 utilized to perform the specificamplification reaction. Nucleotide #27 of SEQ ID NO:12 appears to be thelast nucleotide of a codon triplet, and nucleotides #244-#245 of SEQ IDNO:12 appear to be the first two nucleotides of a codon triplet.Therefore, nucleotides #28 to #243 of SEQ ID NO:12 correspond to apartial coding sequence of mV2. Due to the function of oligonucleotides#6 and #7 in initiating the amplification reaction, they may notcorrespond exactly to the actual sequence encoding the murine BMP-12related protein of the invention and are therefore not translated in thecorresponding amino acid sequence derivation (SEQ ID NO:13).

Oligonucleotide probes designed on the basis of the specificallyamplified murine BMP-12 related DNA sequence set forth in SEQ ID NO:12can be utilized by those skilled in the art to identify full-lengthmurine BMP-12 related encoding clones (either cDNA or genomic).

DNA sequence analysis of another of the resulting subclones named mV9,indicates that the specifically amplified DNA sequence contained thereinencodes a portion of a murine BMP-12 related sequence of the invention.This sequence appears to be the murine homolog to the human MP52 DNAsequence described at SEQ ID NO:3. The DNA sequence (SEQ ID NO:14) andderived amino acid sequence (SEQ ID NO:15) of this specificallyamplified murine DNA fragment are shown in the sequence listings.

Nucleotides #1-#26 of SEQ ID NO:14 comprise a portion of oligonucleotide#6 and nucleotides #246-#272 comprise a portion of the reversecompliment of oligonucleotide #7 utilized to perform the specificamplification reaction. Nucleotide #27 of SEQ ID NO:14 appears to be thelast nucleotide of a codon triplet, and nucleotides #244-#245 of SEQ IDNO:14 appear to be the first two nucleotides of a codon triplet.Therefore, nucleotides #28 to #243 of SEQ ID NO:14 correspond to apartial coding sequence of mV9. Due to the function of oligonucleotides#6 and #7 in initiating the amplification reaction, they may notcorrespond exactly to the actual sequence encoding the murine BMP-12related protein of the invention and are therefore not translated in thecorresponding amino acid sequence derivation (SEQ ID NO:15).

Oligonucleotide probes designed on the basis of the specificallyamplified murine BMP-12 related DNA sequence set forth in SEQ ID NO:14can be utilized by those skilled in the art to identify full-lengthmurine BMP-12 related encoding clones (either cDNA or genomic).

Alternatively, oligonucleotide primers #6 and #7 identified above areutilized as primers to allow the specific amplification of a 275 basepair DNA probe, the internal 259 bp of which correspond to nucleotides#607 to #865 of SEQ ID NO:1, from the BMP-12 encoding plasmid subdlonePCR1-1#2. This 275bp DNA probe was radioactively labelled with ³²P andemployed to screen a murine genomic library constructed in the vector λFIX II (Stratagene catalog #946306). 1 million recombinants of themurine genomic library are plated at a density of approximately 20,000recombinants per plate on 50 plates. Duplicate nitrocellulose replicasof the recombinant bacteriophage plaques are hybridized, under reducedstringency conditions, to the specifically amplified 333 bp probe instandard hybridization buffer (SHB=5×SSC, 0.1% SDS, 5×Denhardt's, 100μg/ml salmon sperm DNA) at 60° C. overnight. The following day theradioactively labelled oligonucleotide containing hybridization solutionis removed an the filters are washed, under reduced stringencyconditions, with 2×SSC, 0.1% SDS at 60° C. Multiple positivelyhybridizing recombinants are identified and plaque purified. Fragmentsof the positively hybridizing murine genomic recombinant clones aresubcloned into standard plasmid vectors (i.e. pGEM-3) and subjected toDNA sequence analysis.

DNA sequence analysis of one of these subclones named MVR3 indicatesthat it encodes a portion of the mouse gene corresponding to the PCRproduct mV1 (murine homolog of the human BMP-12 sequence set forth inSEQ ID NO:1) described above. The partial DNA sequence of this subcloneand corresponding amino acid translation are set forth in SEQ ID NO:29and SEQ ID NO:30 respectively.

DNA sequence analysis of another one of these subclones named MVR32indicates that it encodes a portion of the mouse gene corresponding tothe PCR product mV2 (murine homolog of the human VL-1 sequence set forthin SEQ ID NO:7) described above. The partial DNA sequence of thissubclone and corresponding amino acid translation are set forth in SEQID NO:31 and SEQ ID NO:32 respectively. DNA-sequence analysis of anotherof these subclones named MVR23 indicates that it encodes a portion ofthe mouse gene corresponding to the PCR product mV9 (murine homolog ofthe MP-52 sequence set forth in SEQ ID NO:3) described above.

In a similar manner to that which is described above for identifying andisolating human genomic clones encoding the BMP-12 protein of theinvention, oligonucleotide probe(s) corresponding to the VL-1 encodingsequence set forth in SEQ ID NO:7 can be designed and utilized toidentify human genomic or cDNA sequences encoding the VL-1 (BMP-13)protein. These oligonucleotides would be designed to regions specificfor VL-1 encoding sequences and would therefore be likely to be derivedfrom regions of the lowest degree of nucleotide sequence identitybetween the specifically amplified VL-1 encoding sequence (SEQ ID NO:7)and the specifically amplified BMP-12 encoding sequence (SEQ ID NO:5).

Alternatively, oligonucleotide primers #4 and #5 identified above areutilized as primers to allow the specific amplification of a 333 basepair DNA probe, the internal 315 bp of which correspond to nucleotides#571 to #885 of SEQ ID NO:1, from the BMP-12 encoding plasmid subclonePCRI-1#2. This 333 bp DNA probe was radioactively labelled with ³²P andemployed to screen a human genomic library constructed in the vectorλDASH II (Stratagene catalog #945203). 1 million recombinants of thehuman genomic library are plated at a density of approximately 20,000recombinants per plate on 50 plates. Duplicate nitrocellulose replicasof the recombinant bacteriophage plaques are hybridized, under reducedstringency conditions, to the specifically amplified 333 bp probe instandard hybridization buffer (SHB=5×SSC, 0.1% SDS, 5×Denhardt's, 100μg/ml salmon sperm DNA) at 60° C. overnight. The following day theradioactively labelled oligonucleotide containing hybridization solutionis removed an the filters are washed, under reduced stringencyconditions, with 2×SSC, 0.1% SDS at 60° C. Multiple (approximately 15)positively hybridizing recombinants are identified and plaque purified.

In order to distinguish positively hybridizing recombinants encoding theVL-1 protein of the invention from BMP-12 and other BMP-12-relatedencoding recombinants which would be predicted to hybridize positivelyto the 333 bp DNA probe generated from the BMP-12 encoding plasmidPCR1-1#2 utilized in this screening procedure, the followingoligonucleotide probe, based on the VL-1 sequence set forth in SEQ IDNO:7, is designed and synthesized on an automated DNA synthesizer:

#8: TGTATGCGACTTCCCGC [SEQUENCE ID NO:35]

An oligonucleotide corresponding to nucleotides #60 to #76 of SEQ IDNO:7 which contains 5 nucleotide differences to the corresponding regionof the BMP-12 encoding sequence set forth in SEQ ID NO:1 (nucleotides#672 to #689) One of the recombinant bacteriophage clones whichhybridizes to the VL-1 oligonucleotide probe #8 is designated λJLDc31.This recombinant bacteriophage clone is plaque purified, a bacteriophageplate stock is made and bacteriophage DNA is isolated from the λJLDc31human genomic clone. The bacteriophage λJLDc31 has been deposited withthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. “ATCC” under the accession #75922 on Oct. 20, 1994. This depositmeets the requirements of the Budapest Treaty of the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure and Regulations thereunder. The oligonucleotide hybridizingregion of this recombinant, λJLDc31, is localized to a 2.5 kb EcoRIfragment. This fragment is subcloned into a plasmid vector (pGEM-3) andDNA sequence analysis is performed.

This plasmid subdlone is designated pGEMJLDc31/2.5 and has beendeposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. “ATCC” under the accession # 69710 on Oct. 20,1994. This deposit meets the requirements of the Budapest Treaty of theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and Regulations thereunder.

The partial DNA sequence (SEQ ID NO:25) and derived amino acid sequence(SEQ ID NO:26) of a portion of the 2.5 kb DNA insert of the plasmidsubclone pGEMJLDc31/2.5, derived from clone VJLDc3 1, are shown in theSequence Listings The DNA sequence of a portion of the 2.5 kb EcoRIinsert of the plasmid pGEMJLDc31/2.5 is set forth in SEQ ID NO:25contains an 912 bp open reading frame, as defined by nucleotides #52through #963 of SEQ ID NO:25. As this sequence is derived from a genomicclone it is difficult to determine the boundary between the 5′ extent ofcoding sequence and the 3′ limit of intervening sequence(intron/non-coding sequence). The entire open reading frame (nucleotides#52 through #963 of SEQ ID NO:25) encodes a portion of the VL-1 proteinof the invention of up to 304 amino acids.

Based on the knowledge of other BMP proteins and other proteins withinthe TGF-β family, it is predicted that the precursor polypeptide wouldbe cleaved at the multibasic sequence Arg-Arg-Arg-Arg (SEQ ID NO:26,residues −4 to −1) in agreement with a proposed consensus proteolyticprocessing sequence of Arg-X-X-Arg (SEQ ID NO;26, residues −4 to −1).Cleavage of the VL-1 precursor polypeptide is expected to generate a 120amino acid mature peptide beginning with the amino acid Thr at position#1 of SEQ ID NO:26. The processing of VL-1 into the mature form isexpected to involve dimerization and removal of the N-terminal region ina manner analogous to the processing of the related protein TGF-β[Gentry et al., Molec & Cell. Biol., 8:4162 (1988); Derynck et al.Nature, 316:701 (1985)].

It is contemplated therefore that the mature active species of VL-1comprises a homodimer of two polypeptide subunits, each subunitcomprising amino acids #1 to #120 of SEQ ID NO:26 with a predictedmolecular weight of approximately 12,000 daltons. Further active speciesare contemplated comprising at least amino acids #19 to # 119 or #120 ofSEQ ID NO:26, thereby including the first and last conserved cysteineresidue.

Using such a method, a clone encoding the mature human VL-1 (BMP-13) wasobtained. The nucleotide sequence and corresponding amino acid sequenceencoded by this clone are listed in the Sequence Listings at SEQ IDNO:25 and 26, respectively.

EXAMPLE 2 Expression of BMP-12

In order to produce human BMP-12 proteins, the DNA encoding it istransferred into an appropriate expression vector and introduced intomammalian cells or other preferred eukaryotic or prokaryotic hosts byconventional genetic engineering techniques.

In order to produce the human BMP-12 protein in bacterial cells, thefollowing procedure is employed.

Expression of BMP-12 in E. coli

An expression plasmid pALV1-781, for production of BMP-12 in E. coli wasconstructed which contains the following principal features. Nucleotides1-2060 contain DNA sequences originating from the plasmid pUC-18[Norrander et al., Gene 26:101-106 (1983)] including sequencescontaining the gene for β-lactamase which confers resistance to theantibiotic ampicillin in host E. coli strains, and a colE1-derivedorigin of replication. Nucleotides 2061-2221 contain DNA sequences forthe major leftward promotor (pL) of bacteriophage λ [Sanger et al., J.Mol. Biol. 162:729-773 (1982)], including three operator sequences0_(L)1, 0_(L)2 and 0_(L)3. The operators are the binding sites for λcIrepressor protein, intracellular levels of which control the amount oftranscription initiation from pL. Nucleotides 2222-2723 contain a strongribosome binding sequence included on a sequence derived fromnucleotides 35566 to 35472 and 38137 to 38361 from bacteriophage lambdaas described in Sanger et al., J. Mol. Biol. 162:729-773 (1982).Nucleotides 2724-3041 contain a DNA sequence encoding mature BMP-12protein with all 3′ untranslated sequence removed. The BMP-12 DNAsequences introduced into the pALV1-781 expression vector were modifiedat the 5′ end to raise the A+T content without altering the codingcapacity. These changes were made to increase the fficiency oftranslation initiated on the BMP-12 mRNA in E. coli. Nucleotides3042-3058 provide a “Linker” DNA sequence containing restrictionendonuclease sites. Nucleotides 3059-3127 provide a transcriptiontermination sequence based on that of the E. coli asp A gene [Takagi etal., Nucl. Acids Res. 13:2063-2074 (1985)]. Nucleotides 3128-3532 areDNA sequences derived from pUC-18.

Plasmid pALV1-781 was transformed into the E. coli host strain GI724 (F,lacI^(q), Lacp^(L8), ampC::λcI⁺) by-the procedure of Dagert and Ehrlich,Gene 6:23 (1979). G1724 (ATCC accession No. 55151) contains a copy ofthe wild-type λcI repressor gene stably integrated into the chromosomeat the ampC locus, where it has been placed under the transcriptionalcontrol of Salmonella typhimurium trp promotor/operator sequences. InGI724, λCI protein is made only during growth in tryptophan-free media,such as minimal media or a minimal medium supplemented with casaminoacids such as IMC, described above. Addition of tryptophan to a cultureof GI724 will repress the t1 promoter and turn off synthesis of λcI,gradually causing the induction of transcription from pL promoters ifthey are present in the cell.

Transformants were selected on 1.5% w/v agar plates containing IMCmedium, which is composed of M9 medium [Miller, “Experiments inMolecular Genetics,” Cold Spring Harbor Laboratory, New York (1972)]containing 1 mM MgSO₄ and supplemented with 0.5% w/v glucose, 0.2% w/vcasamino acids and 100 μg/ml ampicillin. GI724 transformed withpALV1-781 was grown at 37° C. to an A₅₅₀ of 0.5 in IMC medium containing100 μg/ml ampicillin. Tryptophan was then added to a final concentrationof 100 μg/ml and the culture incubated for a further 4 hours. Duringthis time BMP-12 protein accumulates within the “inclusion body”fraction.

Preparation of Protein Monomer

18 g of frozen cells were weighed out and resuspended in 60 ml of 100 mMTris, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], pH 8.3.Cells were lysed by 3 passes through a Microfluidizer™ [model #MCF 100T]. The inclusion body pellet was obtained by centrifugation at 15,000 gat 4° C. for 20 minutes. The supernatant was decanted, and the pelletwas washed with 100 ml of 100 mM Tris, 1.0 M NaCl, 10 mM EDTA, 1 mMPMSF, pH 8.3. The suspension was centrifuged again at 15,000 g at 4° C.for 10 minutes, and the supernatant decanted. The pellet was then washedwith 100 ml of 100 mM Tris, 10 mM EDTA, 1% Triton X-100, 1 mM PMSF, pH8.3. The suspension was centrifuged again at 15,000 g at 4° C. for 10minutes, and the supernatant decanted. The pellet was resuspended with50 ml of 20 mM Tris, 1 mM EDTA, 1 mM PMSF, pH 8.3, containing 1% DTT ina glass tissue homogenizer. Monomeric BMP-12 was then solubilized byacidification to pH 2.5 with glacial acetic acid. The soluble fractionwas isolated by centrifugation at 15,000 g for 20 minutes at 4° C.

The supernatant from this centrifugation was collected andchromatographed over a Sephacryl S-100™ size exclusion column (83 cm×2.6cm; ≈440 ml bed) in 20 ml increments. The Sephacryl S-100™ column wasrun with a mobile phase of 1% acetic acid at a flow rate of 1.4 ml/min.Fractions corresponding to BMP-12 monomer were detected by absorbance at280 nm, and using a computer calculated extinction coefficient of18200M⁻¹ cm⁻¹ and molecular weight (11667 daltons). This size exclusioncolumn pooled material was used as starting material for refoldingreactions.

As an alternative to the above, 1.0 g of cells stored at −80° C. aremeasured. Solution (3.4 ml 100 mM TRIS, 10 mM EDTA, pH 8.5) is added.The solution is vortexed until cells are well suspended. 40 μl 100 mMPMSF in isopropanol is added. The cells are lysed at 1000 psi in aFrench pressure cell. The inclusion bodies are centrifuged at 4° C. for20 minutes in an Eppendorf microfuge to form pellets. The supernatantsare decanted. To one pellet (out of 4 total) 1.0 ml degassed 8.0 Mguanidine hydrochloride, 0.5 M TRIS, 5 mM EDTA, pH 8.5, containing 250mM DTT is added. The pellet is dissolved and argon is blown over theliquid for 30 seconds. Next the solution is incubated at 37° C. for onehour. Insoluble material is pelleted for 2-3 minutes in an Eppendorfmicrofuge at 23° C. 0.5-1.0 ml of supernatant is injected onto a Supelco2 cm guard cartridge (LC-304), and eluted with an acetonitrile gradientin 0.1% TFA from 1-70% over 35 minutes. BMP-12 elutes between 29 and 31minutes. Fractions are pooled and the protein concentration determinedby adsorbance at 280 nanometers versus 0.1% TFA, using the theoreticalextinction coefficient based upon the amino acid content.

As a second alternate method to the above, frozen cell pellets obtainedfrom the E. coli transformants as described above are thawed in 30 ml ofTE8.3(100:10) buffer (100 mM Tris-HCl pH 8.3, 10 mM Na₂EDTA, 1 mM PMSF).Cells are lysed by three passes through a Microfluidizer™ [model #MCF100 T]. The initial inclusion body material pellet is dissolved in 8 Mguanidine-HCI, TE8.5(100:10) buffer (100 mM Tris-HCI pH 8.5, 10 mMNa₂EDTA which contained 100 mM DTT, and incubated at 37° C. for 1 hour.This material is centrifuged at 12,000×g for 15 minutes at roomtemperature.

Refolding of BMP-12 Protein Using CHAPS System

A sufficient volume of the BMP-12 pool is lyophilized to give 10 μg ofprotein. 5 μl of glass distilled water is added to redissolve theresidue, then 100 μl of refold mix (50 mM Tris, 1.0 M NaCl, 2%3-(3-chlolamido-propyl)dimethylammonio-1-propane-sulfate (CHAPS), 5 mMEDTA, 2 mM glutathione (reduced) 1 mM glutathione (oxidized); at pH ofapproximately 8.5). The solution is gently mixed and stored at 23° C.for 1-4 days. Dimer formation is assessed by running an aliquot on aNovex 16% tricine gel at 125 volts for 2.5 hours, followed by CoomassieBlue staining and destaining.

BMP-12 dimer was purified using a C4 analytical RP-HPLC (reversedphase-high performance liquid chromatography) column (Vydac 214TP54)which was equilibrated to 1% B buffer (diluted into A buffer) and wasrun over 35 minutes. during which the protein elutes, using thefollowing gradient (A buffer=0.1% trifluoroacetic acid, B buffer=95%acetonitrile, 0.1% trifluoroacetic acid [TFA]), with a flow rate of 1ml/min:

1-5 minutes 20% B buffer

5-10 minutes 20-30% B buffer

10-30 minutes 30 -50% B buffer

30-35 minutes 50-100% B buffer

Protein was monitored by absorbance at 280 nm. Peak BMP-12 fractions(eluting between 29 and 31 minutes) were pooled. Purity was assessed bySDS-PAGE. The concentration was determined by absorbance at 280 nm, andusing the computer calculated extinction coefficient and molecularweight as indicated above.

Expression of BMP-12 in Mammalian Cells

Another contemplated preferred expression system for biologically activerecombinant human BMP-12 is stably transformed mammalian cells.

One skilled in the art can construct mammalian expression vectors byemploying the sequence of SEQ ID NO:1, or other DNA sequences encodingBMP-12 proteins or other modified sequences and known vectors, such aspCD [Okayama et al., Mol. Cell Biol., 2:161-170 (1982)], pJL3, pJL4[Gough et al., EMBO J., 4:645-653 (1985)] and pMT2 CXM.

The mammalian expression vector pMT2 CXM is a derivative of p91023(b)(Wong et al., Science 228:810-815, 1985) differing from the latter inthat it contains the ampicillin resistance gene in place of thetetracycline resistance gene and further contains a XhoI site forinsertion of CDNA clones. The functional elements of pMT2 CXM have beendescribed (Kaufman, R. J., 1985, Proc. Natl. Acad. Sci. USA 82:689-693)and include the adenovirus VA genes, the SV40 origin of replicationincluding the 72 bp enhancer, the adenovirus major late promoterincluding a 5′ splice site and the majority of the adenovirus tripartiteleader sequence present on adenovirus late mRNAs, a 3′ splice acceptorsite, a DHFR insert, the SV40 early polyadenylation site (SV40), andpBR322 sequences needed for propagation in E. coli.

Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF, which hasbeen deposited with the American Type Culture Collection (ATCC),Rockville, Md. (USA) under accession number ATCC 67122. EcoRI digestionexcises the cDNA insert present in pMT2-VWF, yielding pMT2 in linearform which can be ligated and used to transform E. coli HB 101 or DH-5to ampicillin resistance. Plasmid pMT2 DNA can be prepared byconventional methods. pMT2 CXM is then constructed using loopout/inmutagenesis [Morinaga, et al., Biotechnology 84: 636 (1984). Thisremoves bases 1075 to 1145 relative to the Hind III site near the SV40origin of replication and enhancer sequences of pMT2. In addition itinserts a sequence containing the recognition site for the restrictionendonuclease Xho I. A derivative of pMT2CXM, termed pMT23, containsrecognition sites for the restriction endonucleases PstI, Eco RI, Salland Xhol. Plasmid pMT2 CXM and pMT23 DNA may be prepared by conventionalmethods.

pEMC2β1 derived from pMT21 may also be suitable in practice of theinvention. pMT21 is derived from pMT2 which is derived from pMT2-VWF. Asdescribed above EcoRI digestion excises the cDNA insert present inpMT-VWF, yielding pMT2 in linear form which can be ligated and used totransform E. Coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2DNA can be prepared by conventional methods.

pMT21 is derived from pMT2 through the following two modifications.First, 76 bp of the 5′ untranslated region of the DHFR cDNA including astretch of 19 G residues from G/C tailing for cDNA cloning is deleted.In this process, a XhoI site is inserted to obtain the followingsequence immediately upstream from DHFR. Second, a unique Clal site isintroduced by digestion with EcoRV and XbaI, treatment with Klenowfragment of DNA polymerase I, and ligation to a ClaI linker (CATCGATG).This deletes a 250 bp segment from the adenovirus associated RNA (VAI)region but does not interfere with VAI RNA gene expression or function.pMT21 is digested with EcoRI and XhoI, and used to derive the vectorpEMC2B1.

A portion of the EMCV leader is obtained from pMT2-ECAT1 [S. K. Jung, etal, J. Virol 63:1651-1660 (1989)] by digestion with Eco RI and Pstl,resulting in a 2752 bp fragment. This fragment is digested with TaqIyielding an Eco RI-TaqI fragment of 508 bp which is purified byelectrophoresis on low melting agarose gel. A 68 bp adapter and itscomplementary strand are synthesized with a 5′ TaqI protruding end and a3′ XhoI protruding end which has a sequence which matches the EMC virusleader sequence from nucleotide 763 to 827. It also changes the ATG atposition 10 within the EMC virus leader to an ATT and is followed by aXhoI site. A three way ligation of the pMT21 Eco RI-XhoI fragment, theEMC virus EcoRI-TaqI fragment, and the 68 bp oligonucleotide adapterTaqI-XhoI adapter. resulting in the vector pEMC2β1.

This vector contains the SV40 origin of replication and enhancer, theadenovirus major late promoter, a cDNA copy of the majority of theadenovirus tripartite leader sequence, a small hybrid interveningsequence, an SV40 polyadenylation signal and the adenovirus VA I gene,DHFR and β-lactamase markers and an EMC sequence, in appropriaterelationships to direct the high level expression of the desired cDNA inmammalian cells.

The construction of vectors may involve modification of the BMP-12 DNAsequences. For instance, BMP-12 cDNA can be modified by removing thenon-coding nucleotides on the 5′ and 3′ ends of the coding region. Thedeleted non-coding nucleotides may or may not be replaced by othersequences known to be beneficial for expression. These vectors aretransformed into appropriate host cells for expression of BMP-12proteins. Additionally, the sequence of SEQ ID NO:1 or other sequencesencoding BMP-12 proteins can be manipulated to express BMP-12 protein byisolating the mature coding sequence of nucleotides 571 to 882 of SEQ IDNO:1 and adding at the 5′ end sequences encoding the completepropeptides of other BMP proteins.

For example, one skilled in the art can make a fusion protein in whichthe propeptide of BMP-2 is linked in operable fashion to the matureBMP-12 peptide by preparing a DNA vector in which the DNA sequenceencoding the BMP-2 propeptide is linked in proper reading frame to theDNA sequence encoding the mature BMP-12 peptide. The DNA sequence ofsuch a fusion protein is shown in SEQUENCE ID NO:27.

One skilled in the art can manipulate the sequences of SEQ ID NO:1 byeliminating or-replacing the mammalian regulatory sequences flanking thecoding sequence with bacterial sequences to create bacterial vectors forintracellular or extracellular expression by bacterial cells, asdescribed above. As another example, the coding sequences could befurther manipulated (e.g. ligated to other known linkers or modified bydeleting non-coding sequences therefrom or altering nucleotides thereinby other known techniques). The modified BMP-12 coding sequence couldthen be inserted into a known bacterial vector using procedures such asdescribed in T. Taniguchi et al., Proc. Natl Acad. Sci. USA,77:5230-5233 (1980). This exemplary bacterial vector could then betransformed into bacterial host cells and a BMP-12 protein expressedthereby. For a strategy for producing extracellular expression of BMP-12proteins in bacterial cells, see, e.g. European patent application EPA177,343.

Similar manipulations can be performed for the construction of an insectvector [See, e.g. procedures described in published European patentapplication 155,476] for expression in insect cells. A yeast vectorcould also be constructed employing yeast regulatory sequences forintracellular or extracellular expression of the factors of the presentinvention by yeast cells. [See, e.g., procedures described in publishedPCT application WO86/00639 and European patent application EPA 123,289].

A method for producing high levels of a BMP-12 protein of the inventionin mammalian cells may involve the construction of cells containingmultiple copies of the heterologous BMP-12 gene. The heterologous geneis linked to an amplifiable marker, e.g. the dihydrofolate reductase(DHFR) gene for which cells containing increased gene copies can beselected for propagation in increasing concentrations of methotrexate(MTX) according to the procedures of Kaufman and Sharp, J. Mol. Biol.,159:601-629 (1982). This approach can be employed with a number ofdifferent cell types.

For example, a plasmid containing a DNA sequence for a BMP-12 of theinvention in operative association with other plasmid sequences enablingexpression thereof and the DHFR expression plasmid pAdA26SV(A)3 [Kaufmanand Sharp, Mol. Cell. Biol., 2:1304 (1982)] can be co-introduced intoDHFR-deficient CHO cells, DUKX-BII, by various methods including calciumphosphate coprecipitation and transfection, electroporation orprotoplast fusion. DHFR expressing transfornants are selected for growthin alpha media with dialyzed fetal calf serum, and subsequently selectedfor amplification by growth in increasing concentrations of MTX (e.g.sequential steps in 0.02, 0.2, 1.0 and 5 uM MTX) as described in Kaufmanet al., Mol Cell Biol., 5:1750 (1983), Transformants are cloned, andbiologically active BMP-12 expression is monitored by the Rosen-modifiedSampath-Reddi rat assay described below in Example 5. BMP-12 expressionshould increase with increasing levels of MTX resistance. BMP-12polypeptides are characterized using standard techniques known in theart such as pulse labeling with [35S] methionine or cysteine andpolyacrylamide gel electrophoresis. Similar procedures can be followedto produce other related BMP-12 proteins.

EXAMPLE 3 Preparation of BMP-2 Propeptide/BMP-12 Mature Peptide Fusion

In order to construct a vector encoding the BMP-2 propeptide/BMP-12mature peptide fusion, the following cloning procedure was used to fusethe two sequences together.

First, a DNA restriction enzyme fragment comprising the propeptide ofhuman BMP-2 protein, comprising nucleotides 1 through 843 of SEQ IDNO:27 is cut from pBMP2ΔEMC. pBMP2ΔEMC is a plasmid derived from lambdaU20S-39 (ATCC #40345) comprising the entire coding sequence for humanBMP-2 protein with the non-translated 5′ and 3′ sequences of BMP-2deleted from the vector. The 5′ restriction enzyme used was Bgl II andit cuts pBMP2ΔEMC in the vector at nucleotide 979. The 3′ restrictionenzyme used was Mae II and it cuts pBMP2Δ EMC in the BMP-2 propeptide atnucleotide 1925, just short of the carboxy terminus. The resulting 954base pair product was then gel isolated and gene cleaned. Second, a DNArestriction enzyme fragment comprising the 5′ portion of the humanBMP-12 mature peptide DNA sequence, is cut from pPCR1-1#2 V1-1 (ATCC#69517). The 5′ restriction enzyme used was Eae I and it cuts pPCR1-1#2V1-1 just 3′ of N-terminus of the human BMP-12 mature peptide sequence.The resulting 259 base pair product was gel isolated and gene cleaned.Third, two DNA oligos were designed and synthesized, so that whenannealed would form a tiny DNA fragment comprising fusion sequence ofthe extreme 3′ end of the human BMP-2 propeptide and the 5′ end ofBMP-12 mature peptide. The DNA fragment has a 5′ Mae II complimentarysticky end which anneals to the 3′ restriction enzyme fragmentcomprising the human BMP-2 propeptide. The annealed oligo DNA fragmenthas a 3′ Eae I complimentary sticky end which anneals to the 5′ of therestriction enzyme fragment comprising the mature peptide of humanBMP-12. The coding strand oligo is named B2/12 and is 13 base pairslong. Next, a DNA fragment encoding the 123 base pairs at the 3′ end ofthe BMP-12 mature peptide fragment was obtained as follows. First, a DNAfragment comprising the propeptide of human BMP-2 protein, comprisingnucleotides 1 through 846 is PCR amplified from pBMP2ΔEMC. The 5′ primer(oligo 655a) anneals just 5′ of the polylinker. The 3′ primer (BMPpro3)anneals to the BMP-2 propeptide 3′ end and introduces a Bgl IIrestriction enzyme site by silent sequence mutations. The resulting PCRproduct was cut with Sal I, which cleaves in the polylinker, and Bgl II.The 850 base pair restriction enzyme fragment (ending in amino acidsequence REKR) was gel isolated and gene cleaned. The BMP-12 maturepeptide was PCR amplified using a 5′ primer (oligo 5-1) encoding the BglII restriction enzyme site by silent sequence mutations, and annealingto the 5′ end of a possible mature cleavage product, beginning withamino acid sequence SRCS. The 3′ primer (V1-13) anneals to the BMP-12mature peptide 3′ end and introduces a Xba I restriction enzyme siteafter the stop codon. The resulting PCR product was cut with Bgl II andXba I. The 321 base pair restriction enzyme fragment was gel isolatedand gene cleaned.

The two restriction fragments were three-way ligated into a previouslySall and XbaI cut vector. The resultant construct was sequenced to checkfor PCR induced errors and a silent C to T mutation was observed at basepair 185 in the propeptide. This plasmid was designated pREKRSRC. ThenpREKRSRC was cut with BglII and NgoMI, and the vector fragmentencompassing the last 123 base pairs of the BMP12 mature sequence wasthereby isolated. The three restriction fragments and the annealedoligolinker were four-way ligated to yield pREKR-TAL with the BMP-2propeptide with the mature cleavage site at the 3′ end fused to the(TAL) 5′ end of the BMP-12 mature peptide. The coding sequence of theresulting ligated vector is shown in SEQ ID NO:27.

EXAMPLE 4 Biological Activity of Expressed BMP-12

To measure the biological activity of the expressed BMP-12 proteinsobtained in Example 2 above, the proteins are recovered from the cellculture and purified by isolating the BMP-12 proteins from otherproteinaceous materials with which they are co-produced as well as fromother contaminants. The purified protein may be assayed in accordancewith the rat assay described below in Example 5.

Purification is carried out using standard techniques known to thoseskilled in the art.

Protein analysis is conducted using standard techniques such as SDS-PAGEacrylamide [Laemmli, Nature 227:680 (1970)] stained with Coomassie Blueor silver [Oakley, et al. Anal. Biochem. 105:361 (1980)] and byimmunoblot [Towbin, et al. Proc. Nati. Acad. Sci. USA 76:4350 (1979)]

EXAMPLE 5 ROSEN MODIFIED SAMPATH-REDDI ASSAY

A modified version of the rat ectopic implant assay described in Sampathand Reddi, Proc. Natl. Acad. Sci. USA, 80:6591-6595 (1983) is used toevaluate the activity of the BMP-12 proteins. This modified assay isherein called the Rosen-modified Sampath-Reddi assay. The assay has beenwidely used to evaluate the bone and cartilage-inducing activity ofBMPs. The ethanol precipitation step of the Sampath-Reddi procedure isreplaced by dialyzing (if the composition is a solution) or diafiltering(if the composition-is a suspension) the fraction to be assayed againstwater. The solution or suspension is then equilibrated to 0.1% TFA. Theresulting solution is added to 20 mg of rat matrix. A mock rat matrixsample not treated with the protein serves as a control. This materialis frozen and lyophilized and the resulting powder enclosed in #5gelatin capsules. The capsules are implanted subcutaneously in theabdominal thoracic area of 21-49 day old male Long Evans rats. Theimplants are removed after 10 days. A section of each implant is fixedand processed for histological analysis. 1 μm glycolmethacrylatesections are stained with Von Kossa and acid fuschin to score the amountof induced tendon/ligament-like tissue formation present in eachimplant.

BMP-42 was implanted in the rats in doses of 1, 5, 25 and 50 μg perimplant for 10 days. BMP-2 at a dose of 5 μg was included as a positivecontrol. For all doses of BMP-12 tested, no bone or cartilage formationwas observed in the implants after ten days. Instead, the implants werefilled with tissue resembling embryonic tendon, which is easilyrecognized by the presence of dense bundles of fibroblasts oriented inthe same plane and packed tightly together. [Tendon/ligament-like tissueis described, for example, in Ham and Cornack, Histology (JB LippincottCo. (1979), pp. 367-369, the disclosure of which is hereby incorporatedby reference]. These findings were reproduced in a second set of assaysin which tendon/ligament-like tissues was present in all BMP-12containing implants. In contrast, the BMP-2 implants, as expected,showed cartilage and bone formation, but contained notendon/ligament-like tissue.

The BMP-12 proteins and related proteins of this invention may beassessed for activity on this assay.

EXAMPLE 6

Using methods in accordance with the above examples, with minormodifications within the skill of the art, human MP52 protein and themurine homologue of BMP-13 protein were expressed and assayed fortendon/ligament-like tissue inducing activity. All proteins showedcomparable results, similar to those described above for human BMP-12.

The foregoing descriptions detail presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto. Thedisclosure of all references discussed herein are hereby incorporated byreference.

35 926 base pairs nucleic acid single linear DNA (genomic) Homo sapiensv1-1 mat_peptide 571..882 CDS 1..882 1 GCG CGT AAT ACG ACT CAC TAT AGGGCG AAT TGG GTA CGG GGC CCA GGC 48 Ala Arg Asn Thr Thr His Tyr Arg AlaAsn Trp Val Arg Gly Pro Gly -190 -185 -180 -175 AGC TGG ACT TCT CCG CCGTTG CTG CTG CTG TCC ACG TGC CCG GGC GCC 96 Ser Trp Thr Ser Pro Pro LeuLeu Leu Leu Ser Thr Cys Pro Gly Ala -170 -165 -160 GCC CGA GCG CCA CGCCTG CTG TAC TCG CGG GCA GCT GAG CCC CTA GTC 144 Ala Arg Ala Pro Arg LeuLeu Tyr Ser Arg Ala Ala Glu Pro Leu Val -155 -150 -145 GGT CAG CGC TGGGAG GCG TTC GAC GTG GCG GAC GCC ATG AGG CGC CAC 192 Gly Gln Arg Trp GluAla Phe Asp Val Ala Asp Ala Met Arg Arg His -140 -135 -130 CGT CGT GAACCG CGC CCC CCC CGC GCG TTC TGC CTC TTG CTG CGC GCA 240 Arg Arg Glu ProArg Pro Pro Arg Ala Phe Cys Leu Leu Leu Arg Ala -125 -120 -115 GTG GCAGGC CCG GTG CCG AGC CCG TTG GCA CTG CGG CGA CTG GGC TTC 288 Val Ala GlyPro Val Pro Ser Pro Leu Ala Leu Arg Arg Leu Gly Phe -110 -105 -100 -95GGC TGG CCG GGC GGA GGG GGC TCT GCG GCA GAG GAG CGC GCG GTG CTA 336 GlyTrp Pro Gly Gly Gly Gly Ser Ala Ala Glu Glu Arg Ala Val Leu -90 -85 -80GTC GTC TCC TCC CGC ACG CAG AGG AAA GAG AGC TTA TTC CGG GAG ATC 384 ValVal Ser Ser Arg Thr Gln Arg Lys Glu Ser Leu Phe Arg Glu Ile -75 -70 -65CGC GCC CAG GCC CGC GCG CTC GGG GCC GCT CTG GCC TCA GAG CCG CTG 432 ArgAla Gln Ala Arg Ala Leu Gly Ala Ala Leu Ala Ser Glu Pro Leu -60 -55 -50CCC GAC CCA GGA ACC GGC ACC GCG TCG CCA AGG GCA GTC ATT GGC GGC 480 ProAsp Pro Gly Thr Gly Thr Ala Ser Pro Arg Ala Val Ile Gly Gly -45 -40 -35CGC AGA CGG AGG AGG ACG GCG TTG GCC GGG ACG CGG ACA GCG CAG GGC 528 ArgArg Arg Arg Arg Thr Ala Leu Ala Gly Thr Arg Thr Ala Gln Gly -30 -25 -20-15 AGC GGC GGG GGC GCG GGC CGG GGC CAC GGG CGC AGG GGC CGG AGC CGC 576Ser Gly Gly Gly Ala Gly Arg Gly His Gly Arg Arg Gly Arg Ser Arg -10 -5 1TGC AGC CGC AAG CCG TTG CAC GTG GAC TTC AAG GAG CTC GGC TGG GAC 624 CysSer Arg Lys Pro Leu His Val Asp Phe Lys Glu Leu Gly Trp Asp 5 10 15 GACTGG ATC ATC GCG CCG CTG GAC TAC GAG GCG TAC CAC TGC GAG GGC 672 Asp TrpIle Ile Ala Pro Leu Asp Tyr Glu Ala Tyr His Cys Glu Gly 20 25 30 CTT TGCGAC TTC CCT TTG CGT TCG CAC CTC GAG CCC ACC AAC CAT GCC 720 Leu Cys AspPhe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala 35 40 45 50 ATC ATTCAG ACG CTG CTC AAC TCC ATG GCA CCA GAC GCG GCG CCG GCC 768 Ile Ile GlnThr Leu Leu Asn Ser Met Ala Pro Asp Ala Ala Pro Ala 55 60 65 TCC TGC TGTGTG CCA GCG CGC CTC AGC CCC ATC AGC ATC CTC TAC ATC 816 Ser Cys Cys ValPro Ala Arg Leu Ser Pro Ile Ser Ile Leu Tyr Ile 70 75 80 GAC GCC GCC AACAAC GTT GTC TAC AAG CAA TAC GAG GAC ATG GTG GTG 864 Asp Ala Ala Asn AsnVal Val Tyr Lys Gln Tyr Glu Asp Met Val Val 85 90 95 GAG GCC TGC GGC TGCAGG TAGCGCGCGG GCCGGGGAGG GGGCAGCCAC 912 Glu Ala Cys Gly Cys Arg 100GCGGCCGAGG ATCC 926 294 amino acids amino acid linear protein notprovided 2 Ala Arg Asn Thr Thr His Tyr Arg Ala Asn Trp Val Arg Gly ProGly -190 -185 -180 -175 Ser Trp Thr Ser Pro Pro Leu Leu Leu Leu Ser ThrCys Pro Gly Ala -170 -165 -160 Ala Arg Ala Pro Arg Leu Leu Tyr Ser ArgAla Ala Glu Pro Leu Val -155 -150 -145 Gly Gln Arg Trp Glu Ala Phe AspVal Ala Asp Ala Met Arg Arg His -140 -135 -130 Arg Arg Glu Pro Arg ProPro Arg Ala Phe Cys Leu Leu Leu Arg Ala -125 -120 -115 Val Ala Gly ProVal Pro Ser Pro Leu Ala Leu Arg Arg Leu Gly Phe -110 -105 -100 -95 GlyTrp Pro Gly Gly Gly Gly Ser Ala Ala Glu Glu Arg Ala Val Leu -90 -85 -80Val Val Ser Ser Arg Thr Gln Arg Lys Glu Ser Leu Phe Arg Glu Ile -75 -70-65 Arg Ala Gln Ala Arg Ala Leu Gly Ala Ala Leu Ala Ser Glu Pro Leu -60-55 -50 Pro Asp Pro Gly Thr Gly Thr Ala Ser Pro Arg Ala Val Ile Gly Gly-45 -40 -35 Arg Arg Arg Arg Arg Thr Ala Leu Ala Gly Thr Arg Thr Ala GlnGly -30 -25 -20 -15 Ser Gly Gly Gly Ala Gly Arg Gly His Gly Arg Arg GlyArg Ser Arg -10 -5 1 Cys Ser Arg Lys Pro Leu His Val Asp Phe Lys Glu LeuGly Trp Asp 5 10 15 Asp Trp Ile Ile Ala Pro Leu Asp Tyr Glu Ala Tyr HisCys Glu Gly 20 25 30 Leu Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro ThrAsn His Ala 35 40 45 50 Ile Ile Gln Thr Leu Leu Asn Ser Met Ala Pro AspAla Ala Pro Ala 55 60 65 Ser Cys Cys Val Pro Ala Arg Leu Ser Pro Ile SerIle Leu Tyr Ile 70 75 80 Asp Ala Ala Asn Asn Val Val Tyr Lys Gln Tyr GluAsp Met Val Val 85 90 95 Glu Ala Cys Gly Cys Arg 100 1207 base pairsnucleic acid single linear DNA (genomic) Homo sapiens MP52 CDS 845..12043 ACCGGGCGGC CCTGAACCCA AGCCAGGACA CCCTCCCCAA ACAAGGCAGG CTACAGCCCG 60GACTGTGACC CCAAAAGGAC AGCTTCCCGG AGGCAAGGCA CCCCCAAAAG CAGGATCTGT 120CCCCAGCTCC TTCCTGCTGA AGAAGGCCAG GGAGCCCGGG CCCCCACGAG AGCCCAAGGA 180GCCGTTTCGC CCACCCCCCA TCACACCCCA CGAGTACATG CTCTCGCTGT ACAGGACGCT 240GTCCGATGCT GACAGAAAGG GAGGCAACAG CAGCGTGAAG TTGGAGGCTG GCCTGGCCAA 300CACCATCACC AGCTTTATTG ACAAAGGGCA AGATGACCGA GGTCCCGTGG TCAGGAAGCA 360GAGGTACGTG TTTGACATTA GTGCCCTGGA GAAGGATGGG CTGCTGGGGG CCGAGCTCCG 420GATCTTGCGG AAGAAGCCCT CGGACACGGC CAAGCCAGCG GCCCCCGGAG GCGGGCGGGC 480TGCCCAGCTG AAGCTGTCCA GCTGCCCCAG CGGCCGGCAG CCGGCCTCCT TGCTGGATGT 540GCGCTCCGTG CCAGGCCTGG ACGGATCTGG CTGGGAGGTG TTCGACATCT GGAAGCTCTT 600CCGAAACTTT AAGAACTCGG CCCAGCTGTG CCTGGAGCTG GAGGCCTGGG AACGGGGCAG 660GGCCGTGGAC CTCCGTGGCC TGGGCTTCGA CCGCGCCGCC CGGCAGGTCC ACGAGAAGGC 720CCTGTTCCTG GTGTTTGGCC GCACCAAGAA ACGGGACCTG TTCTTTAATG AGATTAAGGC 780CCGCTCTGGC CAGGACGATA AGACCGTGTA TGAGTACCTG TTCAGCCAGC GGCGAAAACG 840GCGG GCC CCA CTG GCC ACT CGC CAG GGC AAG CGA CCC AGC AAG AAC CTT 889 AlaPro Leu Ala Thr Arg Gln Gly Lys Arg Pro Ser Lys Asn Leu 1 5 10 15 AAGGCT CGC TGC AGT CGG AAG GCA CTG CAT GTC AAC TTC AAG GAC ATG 937 Lys AlaArg Cys Ser Arg Lys Ala Leu His Val Asn Phe Lys Asp Met 20 25 30 GGC TGGGAC GAC TGG ATC ATC GCA CCC CTT GAG TAC GAG GCT TTC CAC 985 Gly Trp AspAsp Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Phe His 35 40 45 TGC GAG GGGCTG TGC GAG TTC CCA TTG CGC TCC CAC CTG GAG CCC ACG 1033 Cys Glu Gly LeuCys Glu Phe Pro Leu Arg Ser His Leu Glu Pro Thr 50 55 60 AAT CAT GCA GTCATC CAG ACC CTG ATG AAC TCC ATG GAC CCC GAG TCC 1081 Asn His Ala Val IleGln Thr Leu Met Asn Ser Met Asp Pro Glu Ser 65 70 75 ACA CCA CCC ACC TGCTGT GTG CCC ACG CGG CTG AGT CCC ATC AGC ATC 1129 Thr Pro Pro Thr Cys CysVal Pro Thr Arg Leu Ser Pro Ile Ser Ile 80 85 90 95 CTC TTC ATT GAC TCTGCC AAC AAC GTG GTG TAT AAG CAG TAT GAG GAC 1177 Leu Phe Ile Asp Ser AlaAsn Asn Val Val Tyr Lys Gln Tyr Glu Asp 100 105 110 ATG GTC GTG GAG TCGTGT GGC TGC AGG TAG 1207 Met Val Val Glu Ser Cys Gly Cys Arg 115 120 120amino acids amino acid linear protein not provided 4 Ala Pro Leu Ala ThrArg Gln Gly Lys Arg Pro Ser Lys Asn Leu Lys 1 5 10 15 Ala Arg Cys SerArg Lys Ala Leu His Val Asn Phe Lys Asp Met Gly 20 25 30 Trp Asp Asp TrpIle Ile Ala Pro Leu Glu Tyr Glu Ala Phe His Cys 35 40 45 Glu Gly Leu CysGlu Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn 50 55 60 His Ala Val IleGln Thr Leu Met Asn Ser Met Asp Pro Glu Ser Thr 65 70 75 80 Pro Pro ThrCys Cys Val Pro Thr Arg Leu Ser Pro Ile Ser Ile Leu 85 90 95 Phe Ile AspSer Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met 100 105 110 Val ValGlu Ser Cys Gly Cys Arg 115 120 128 base pairs nucleic acid singlelinear DNA (genomic) Homo Sapiens V1-1 fragment CDS 28..102 5 GGATCCTGGAAGGATTGGAT CATTGCG CCG CTG GAC TAC GAG GCG TAC CAC 51 Pro Leu Asp TyrGlu Ala Tyr His 1 5 TGC GAG GGC CTT TGC GAC TTC CCT TTG CGT TCG CAC CTCGAG CCC ACC 99 Cys Glu Gly Leu Cys Asp Phe Pro Leu Arg Ser His Leu GluPro Thr 10 15 20 AAC CACGCTATAG TCCAAACCTT TCTAGA 128 Asn 25 25 aminoacids amino acid linear protein not provided 6 Pro Leu Asp Tyr Glu AlaTyr His Cys Glu Gly Leu Cys Asp Phe Pro 1 5 10 15 Leu Arg Ser His LeuGlu Pro Thr Asn 20 25 128 base pairs nucleic acid single linear DNA(genomic) Homo Sapiens VL-1 CDS 28..102 7 GGATCCTGGG ATGACTGGAT TATGGCGCCG CTG GAC TAC GAG GCG TAC CAC 51 Pro Leu Asp Tyr Glu Ala Tyr His 1 5TGC GAG GGT GTA TGC GAC TTC CCG CTG CGC TCG CAC CTG GAG CCC ACC 99 CysGlu Gly Val Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr 10 15 20 AACCACGCCATGC TACAAACGCT TCTAGA 128 Asn 25 25 amino acids amino acid linearprotein not provided 8 Pro Leu Asp Tyr Glu Ala Tyr His Cys Glu Gly ValCys Asp Phe Pro 1 5 10 15 Leu Arg Ser His Leu Glu Pro Thr Asn 20 25 3585base pairs nucleic acid single linear DNA (genomic) not providedpALV1-781 9 CTAACTACCC AACTCAAAAA AAAAAAAAAA AAAAACCCCC TCTAACCCCCATTGACGAAA 60 GGGCCTCGTG ATACGCCTAT TTTTATAGGT TAATGTCATG ATAATAATGGTTTCTTAGAC 120 GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTATTTTTCTAAAT 180 ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA TAAATGCTTCAATAATATTG 240 AAAAAGGAAG AGTATGAGTA TTCAACATTT CCGTGTCGCC CTTATTCCCTTTTTTGCGGC 300 ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG AAAGTAAAAGATGCTGAAGA 360 TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC AACAGCGGTAAGATCCTTGA 420 GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT TTTAAAGTTCTGCTATGTGG 480 CGCGGTATTA TCCCGTATTG ACGCCGGGCA AGAGCAACTC GGTCGCCGCATACACTATTC 540 TCAGAATGAC TTGGTTGAGT ACTCACCAGT CACAGAAAAG CATCTTACGGATGGCATGAC 600 AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT AACACTGCGGCCAACTTACT 660 TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT TTGCACAACATGGGGGATCA 720 TGTAACTCGC CTTGATCGTT GGGAACCGGA GCTGAATGAA GCCATACCAAACGACGAGCG 780 TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC AAACTATTAACTGGCGAACT 840 ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG GAGGCGGATAAAGTTGCAGG 900 ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT GCTGATAAATCTGGAGCCGG 960 TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA GATGGTAAGCCCTCCCGTAT 1020 CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT GAACGAAATAGACAGATCGC 1080 TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA GACCAAGTTTACTCATATAT 1140 ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG ATCTAGGTGAAGATCCTTTT 1200 TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG TTCCACTGAGCGTCAGACCC 1260 CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT CTGCGCGTAATCTGCTGCTT 1320 GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG CCGGATCAAGAGCTACCAAC 1380 TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA CCAAATACTGTCCTTCTAGT 1440 GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA CCGCCTACATACCTCGCTCT 1500 GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG TCGTGTCTTACCGGGTTGGA 1560 CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC TGAACGGGGGGTTCGTGCAC 1620 ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA TACCTACAGCGTGAGCATTG 1680 AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG TATCCGGTAAGCGGCAGGGT 1740 CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC GCCTGGTATCTTTATAGTCC 1800 TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGTCAGGGGGGCG 1860 GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG TTCCTGGCCTTTTGCTGGCC 1920 TTTTGCTCAC ATGTTCTTTC CTGCGTTATC CCCTGATTCT GTGGATAACCGTATTACCGC 1980 CTTTGAGTGA GCTGATACCG CTCGCCGCAG CCGAACGACC GAGCGCAGCGAGTCAGTGAG 2040 CGAGGAAGCG GAAGAGCGCC CAATACGCAA ACCGCCTCTC CCCGCGCGTTGGCCGATTCA 2100 TTAATGCAGA ATTGATCTCT CACCTACCAA ACAATGCCCC CCTGCAAAAAATAAATTCAT 2160 ATAAAAAACA TACAGATAAC CATCTGCGGT GATAAATTAT CTCTGGCGGTGTTGACATAA 2220 ATACCACTGG CGGTGATACT GAGCACATCA GCAGGACGCA CTGACCACCATGAAGGTGAC 2280 GCTCTTAAAA ATTAAGCCCT GAAGAAGGGC AGCATTCAAA GCAGAAGGCTTTGGGGTGTG 2340 TGATACGAAA CGAAGCATTG GCCGTAAGTG CGATTCCGGA TTAGCTGCCAATGTGCCAAT 2400 CGCGGGGGGT TTTCGTTCAG GACTACAACT GCCACACACC ACCAAAGCTAACTGACAGGA 2460 GAATCCAGAT GGATGCACAA ACACGCCGCC GCGAACGTCG CGCAGAGAAACAGGCTCAAT 2520 GGAAAGCAGC AAATCCCCTG TTGGTTGGGG TAAGCGCAAA ACCAGTTCCGAAAGATTTTT 2580 TTAACTATAA ACGCTGATGG AAGCGTTTAT GCGGAAGAGG TAAAGCCCTTCCCGAGTAAC 2640 AAAAAAACAA CAGCATAAAT AACCCCGCTC TTACACATTC CAGCCCTGAAAAAGGGCATC 2700 AAATTAAACC ACACCTATGG TGTATGCATT TATTTGCATA CATTCAATCAATTGTTATCT 2760 AAGGAAATAC TTACATATGT CTCGTTGTTC TCGTAAACCA CTGCATGTAGATTTTAAAGA 2820 GCTCGGCTGG GACGACTGGA TCATCGCGCC GCTGGACTAC GAGGCGTACCACTGCGAGGG 2880 CCTTTGCGAC TTCCCTTTGC GTTCGCACCT CGAGCCCACC AACCATGCCATCATTCAGAC 2940 GCTGCTCAAC TCCATGGCAC CAGACGCGGC GCCGGCCTCC TGCTGTGTGCCAGCGCGCCT 3000 CAGCCCCATC AGCATCCTCT ACATCGACGC CGCCAACAAC GTTGTCTACAAGCAATACGA 3060 GGACATGGTG GTGGAGGCCT GCGGCTGCAG GTAGTCTAGA GTCGACCTGCAGTAATCGTA 3120 CAGGGTAGTA CAAATAAAAA AGGCACGTCA GATGACGTGC CTTTTTTCTTGTGAGCAGTA 3180 AGCTTGGCAC TGGCCGTCGT TTTACAACGT CGTGACTGGG AAAACCCTGGCGTTACCCAA 3240 CTTAATCGCC TTGCAGCACA TCCCCCTTTC GCCAGCTGGC GTAATAGCGAAGAGGCCCGC 3300 ACCGATCGCC CTTCCCAACA GTTGCGCAGC CTGAATGGCG AATGGCGCCTGATGCGGTAT 3360 TTTCTCCTTA CGCATCTGTG CGGTATTTCA CACCGCATAT ATGGTGCACTCTCAGTACAA 3420 TCTGCTCTGA TGCCGCATAG TTAAGCCAGC CCCGACACCC GCCAACACCCGCTGACGCGC 3480 CCTGACGGGC TTGTCTGCTC CCGGCATCCG CTTACAGACA AGCTGTGACCGTCTCCGGGA 3540 GCTGCATGTG TCAGAGGTTT TCACCGTCAT CACCGAAACG CGCGA 3585272 base pairs nucleic acid single linear DNA (genomic) mouse mV1 CDS28..243 10 GGATCCAAGG AGCTCGGCTG GGACGAC TGG ATC ATC GCG CCA TTA GAC TAC51 Trp Ile Ile Ala Pro Leu Asp Tyr 1 5 GAG GCA TAC CAC TGC GAG GGC GTTTGC GAC TTT CCT CTG CGC TCG CAC 99 Glu Ala Tyr His Cys Glu Gly Val CysAsp Phe Pro Leu Arg Ser His 10 15 20 CTG GAG CCT ACC AAC CAC GCC ATC ATTCAG ACG CTG CTC AAC TCC ATG 147 Leu Glu Pro Thr Asn His Ala Ile Ile GlnThr Leu Leu Asn Ser Met 25 30 35 40 GCG CCC GAC GCT GCG CCA GCC TCC TGCTGC GTG CCC GCA AGG CTC AGT 195 Ala Pro Asp Ala Ala Pro Ala Ser Cys CysVal Pro Ala Arg Leu Ser 45 50 55 CCC ATC AGC ATT CTC TAC ATC GAT GCC GCCAAC AAC GTG GTC TAC AAG 243 Pro Ile Ser Ile Leu Tyr Ile Asp Ala Ala AsnAsn Val Val Tyr Lys 60 65 70 CAATACGAGG ACATGGTGGT GGGGAATTC 272 72amino acids amino acid linear protein not provided 11 Trp Ile Ile AlaPro Leu Asp Tyr Glu Ala Tyr His Cys Glu Gly Val 1 5 10 15 Cys Asp PhePro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala Ile 20 25 30 Ile Gln ThrLeu Leu Asn Ser Met Ala Pro Asp Ala Ala Pro Ala Ser 35 40 45 Cys Cys ValPro Ala Arg Leu Ser Pro Ile Ser Ile Leu Tyr Ile Asp 50 55 60 Ala Ala AsnAsn Val Val Tyr Lys 65 70 272 base pairs nucleic acid single linear DNA(genomic) mouse mV2 CDS 28..243 12 GGATCCAAGG AGCTCGGCTG GGACGAC TGG ATTATC GCG CCC CTA GAG TAC 51 Trp Ile Ile Ala Pro Leu Glu Tyr 1 5 GAG GCCTAT CAC TGC GAG GGC GTG TGC GAC TTT CCG CTG CGC TCG CAC 99 Glu Ala TyrHis Cys Glu Gly Val Cys Asp Phe Pro Leu Arg Ser His 10 15 20 CTT GAG CCCACT AAC CAT GCC ATC ATT CAG ACG CTG ATG AAC TCC ATG 147 Leu Glu Pro ThrAsn His Ala Ile Ile Gln Thr Leu Met Asn Ser Met 25 30 35 40 GAC CCG GGCTCC ACC CCG CCT AGC TGC TGC GTT CCC ACC AAA CTG ACT 195 Asp Pro Gly SerThr Pro Pro Ser Cys Cys Val Pro Thr Lys Leu Thr 45 50 55 CCC ATT AGC ATCCTG TAC ATC GAC GCG GGC AAT AAT GTA GTC TAC AAG 243 Pro Ile Ser Ile LeuTyr Ile Asp Ala Gly Asn Asn Val Val Tyr Lys 60 65 70 CAATACGAGGACATGGTGGT GGGGAATTC 272 72 amino acids amino acid linear protein notprovided 13 Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Tyr His Cys Glu GlyVal 1 5 10 15 Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn HisAla Ile 20 25 30 Ile Gln Thr Leu Met Asn Ser Met Asp Pro Gly Ser Thr ProPro Ser 35 40 45 Cys Cys Val Pro Thr Lys Leu Thr Pro Ile Ser Ile Leu TyrIle Asp 50 55 60 Ala Gly Asn Asn Val Val Tyr Lys 65 70 272 base pairsnucleic acid single linear DNA (genomic) mouse mV9 CDS 28..243 14GGATCCAAGG AGCTCGGCTG GGACGAC TGG ATC ATC GCA CCT CTT GAG TAT 51 Trp IleIle Ala Pro Leu Glu Tyr 1 5 GAG GCC TTC CAC TGC GAA GGA CTG TGT GAG TTCCCC TTG CGC TCC CAC 99 Glu Ala Phe His Cys Glu Gly Leu Cys Glu Phe ProLeu Arg Ser His 10 15 20 TTG GAG CCC ACA AAC CAC GCA GTC ATT CAG ACC CTAATG AAC TCT ATG 147 Leu Glu Pro Thr Asn His Ala Val Ile Gln Thr Leu MetAsn Ser Met 25 30 35 40 GAC CCT GAA TCC ACA CCA CCC ACT TGT TGT GTG CCTACA CGG CTG AGT 195 Asp Pro Glu Ser Thr Pro Pro Thr Cys Cys Val Pro ThrArg Leu Ser 45 50 55 CCT ATT AGC ATC CTC TTC ATC GAC TCT GCC AAC AAC GTGGTG TAT AAA 243 Pro Ile Ser Ile Leu Phe Ile Asp Ser Ala Asn Asn Val ValTyr Lys 60 65 70 CAATACGAGG ACATGGTGGT GGGGAATTC 272 72 amino acidsamino acid linear protein not provided 15 Trp Ile Ile Ala Pro Leu GluTyr Glu Ala Phe His Cys Glu Gly Leu 1 5 10 15 Cys Glu Phe Pro Leu ArgSer His Leu Glu Pro Thr Asn His Ala Val 20 25 30 Ile Gln Thr Leu Met AsnSer Met Asp Pro Glu Ser Thr Pro Pro Thr 35 40 45 Cys Cys Val Pro Thr ArgLeu Ser Pro Ile Ser Ile Leu Phe Ile Asp 50 55 60 Ser Ala Asn Asn Val ValTyr Lys 65 70 7 amino acids amino acid single linear peptideBMP/TGF-beta consensus sequence 16 Trp Xaa Asp Trp Ile Xaa Ala 1 5 27base pairs nucleic acid single linear DNA (genomic) not providedoligonucleotide #1 17 CGGATCCTGG VANGAYTGGA THRTNGC 27 6 amino acidsamino acid single linear peptide not provided BMP/TGF-beta consensussequence 18 His Ala Ile Xaa Gln Thr 1 5 28 base pairs nucleic acidsingle linear DNA (genomic) not provided oligonucleotide #2 19TTTCTAGAAR NGTYTGNACD ATNGCRTG 28 40 base pairs nucleic acid singlelinear DNA (genomic) not provided oligonucleotide #3 20 CCACTGCGAGGGCCTTTGCG ACTTCCCTTT GCGTTCGCAC 40 29 base pairs nucleic acid singlelinear DNA (genomic) not provided oligonucleotide #4 21 TGCGGATCCAGCCGCTGCAG CCGCAAGCC 29 29 base pairs nucleic acid single linear DNA(genomic) not provided oligonucleotide #5 22 GACTCTAGAC TACCTGCAGCCGCAGGCCT 29 28 base pairs nucleic acid single linear DNA (genomic) notprovided oligonucleotide #6 23 GCGGATCCAA GGAGCTCGGC TGGGACGA 28 28 basepairs nucleic acid single linear DNA (genomic) not providedoligonucleotide #7 24 GGAATTCCCC ACCACCATGT CCTCGTAT 28 1171 base pairsnucleic acid single linear DNA (genomic) not provided Human VL-1 proteinCDS 2..964 mat_peptide 605..964 25 G AAT TCG GAT CTC TCG CAC ACT CCT CTCCGG AGA CAG AAG TAT TTG 46 Asn Ser Asp Leu Ser His Thr Pro Leu Arg ArgGln Lys Tyr Leu -201-200 -195 -190 TTT GAT GTG TCC ATG CTC TCA GAC AAAGAA GAG CTG GTG GGC GCG GAG 94 Phe Asp Val Ser Met Leu Ser Asp Lys GluGlu Leu Val Gly Ala Glu -185 -180 -175 CTG CGG CTC TTT CGC CAG GCG CCCTCA GCG CCC TGG GGG CCA CCA GCC 142 Leu Arg Leu Phe Arg Gln Ala Pro SerAla Pro Trp Gly Pro Pro Ala -170 -165 -160 -155 GGG CCG CTC CAC GTG CAGCTC TTC CCT TGC CTT TCG CCC CTA CTG CTG 190 Gly Pro Leu His Val Gln LeuPhe Pro Cys Leu Ser Pro Leu Leu Leu -150 -145 -140 GAC GCG CGG ACC CTGGAC CCG CAG GGG GCG CCG CCG GCC GGC TGG GAA 238 Asp Ala Arg Thr Leu AspPro Gln Gly Ala Pro Pro Ala Gly Trp Glu -135 -130 -125 GTC TTC GAC GTGTGG CAG GGC CTG CGC CAC CAG CCC TGG AAG CAG CTG 286 Val Phe Asp Val TrpGln Gly Leu Arg His Gln Pro Trp Lys Gln Leu -120 -115 -110 TGC TTG GAGCTG CGG GCC GCA TGG GGC GAG CTG GAC GCC GGG GAG GCC 334 Cys Leu Glu LeuArg Ala Ala Trp Gly Glu Leu Asp Ala Gly Glu Ala -105 -100 -95 GAG GCGCGC GCG CGG GGA CCC CAG CAA CCG CCG CCC CCG GAC CTG CGG 382 Glu Ala ArgAla Arg Gly Pro Gln Gln Pro Pro Pro Pro Asp Leu Arg -90 -85 -80 -75 AGTCTG GGC TTC GGC CGG AGG GTG CGG CCT CCC CAG GAG CGG GCC CTG 430 Ser LeuGly Phe Gly Arg Arg Val Arg Pro Pro Gln Glu Arg Ala Leu -70 -65 -60 CTGGTG GTA TTC ACC AGA TCC CAG CGC AAG AAC CTG TTC GCA GAG ATG 478 Leu ValVal Phe Thr Arg Ser Gln Arg Lys Asn Leu Phe Ala Glu Met -55 -50 -45 CGCGAG CAG CTG GGC TCG GCC GAG GCT GCG GGC CCG GGC GCG GGC GCC 526 Arg GluGln Leu Gly Ser Ala Glu Ala Ala Gly Pro Gly Ala Gly Ala -40 -35 -30 GAGGGG TCG TGG CCG CCG CCG TCG GGC GCC CCG GAT GCC AGG CCT TGG 574 Glu GlySer Trp Pro Pro Pro Ser Gly Ala Pro Asp Ala Arg Pro Trp -25 -20 -15 CTGCCC TCG CCC GGC CGC CGG CGG CGG CGC ACG GCC TTC GCC AGT CGC 622 Leu ProSer Pro Gly Arg Arg Arg Arg Arg Thr Ala Phe Ala Ser Arg -10 -5 1 5 CATGGC AAG CGG CAC GGC AAG AAG TCC AGG CTA CGC TGC AGC AAG AAG 670 His GlyLys Arg His Gly Lys Lys Ser Arg Leu Arg Cys Ser Lys Lys 10 15 20 CCC CTGCAC GTG AAC TTC AAG GAG CTG GGC TGG GAC GAC TGG ATT ATC 718 Pro Leu HisVal Asn Phe Lys Glu Leu Gly Trp Asp Asp Trp Ile Ile 25 30 35 GCG CCC CTGGAG TAC GAG GCC TAT CAC TGC GAG GGT GTA TGC GAC TTC 766 Ala Pro Leu GluTyr Glu Ala Tyr His Cys Glu Gly Val Cys Asp Phe 40 45 50 CCG CTG CGC TCGCAC CTG GAG CCC ACC AAC CAC GCC ATC ATC CAG ACG 814 Pro Leu Arg Ser HisLeu Glu Pro Thr Asn His Ala Ile Ile Gln Thr 55 60 65 70 CTG ATG AAC TCCATG GAC CCC GGC TCC ACC CCG CCC AGC TGC TGC GTG 862 Leu Met Asn Ser MetAsp Pro Gly Ser Thr Pro Pro Ser Cys Cys Val 75 80 85 CCC ACC AAA TTG ACTCCC ATC AGC ATT CTA TAC ATC GAC GCG GGC AAT 910 Pro Thr Lys Leu Thr ProIle Ser Ile Leu Tyr Ile Asp Ala Gly Asn 90 95 100 AAT GTG GTC TAC AAGCAG TAC GAG GAC ATG GTG GTG GAG TCG TGC GGC 958 Asn Val Val Tyr Lys GlnTyr Glu Asp Met Val Val Glu Ser Cys Gly 105 110 115 TGC AGG TAGCGGTGCCTTTCCCGCCG CCTTGGCCCG GAACCAAGGT GGGCCAAGGT 1014 Cys Arg 120 CCGCCTTGCAGGGGAGGCCT GGCTGCAGAG AGGCGGAGGA GGAAGCTGGC GCTGGGGGAG 1074 GCTGAGGGTGAGGGAACAGC CTGGATGTGA GAGCCGGTGG GAGAGAAGGG AGCGCACCTT 1134 CCCAGTAACTTCTACCTGCC AGCCCAGAGG GAAATAT 1171 321 amino acids amino acid linearprotein not provided 26 Asn Ser Asp Leu Ser His Thr Pro Leu Arg Arg GlnLys Tyr Leu Phe -201 -200 -195 -190 Asp Val Ser Met Leu Ser Asp Lys GluGlu Leu Val Gly Ala Glu Leu -185 -180 -175 -170 Arg Leu Phe Arg Gln AlaPro Ser Ala Pro Trp Gly Pro Pro Ala Gly -165 -160 -155 Pro Leu His ValGln Leu Phe Pro Cys Leu Ser Pro Leu Leu Leu Asp -150 -145 -140 Ala ArgThr Leu Asp Pro Gln Gly Ala Pro Pro Ala Gly Trp Glu Val -135 -130 -125Phe Asp Val Trp Gln Gly Leu Arg His Gln Pro Trp Lys Gln Leu Cys -120-115 -110 Leu Glu Leu Arg Ala Ala Trp Gly Glu Leu Asp Ala Gly Glu AlaGlu -105 -100 -95 -90 Ala Arg Ala Arg Gly Pro Gln Gln Pro Pro Pro ProAsp Leu Arg Ser -85 -80 -75 Leu Gly Phe Gly Arg Arg Val Arg Pro Pro GlnGlu Arg Ala Leu Leu -70 -65 -60 Val Val Phe Thr Arg Ser Gln Arg Lys AsnLeu Phe Ala Glu Met Arg -55 -50 -45 Glu Gln Leu Gly Ser Ala Glu Ala AlaGly Pro Gly Ala Gly Ala Glu -40 -35 -30 Gly Ser Trp Pro Pro Pro Ser GlyAla Pro Asp Ala Arg Pro Trp Leu -25 -20 -15 -10 Pro Ser Pro Gly Arg ArgArg Arg Arg Thr Ala Phe Ala Ser Arg His -5 1 5 Gly Lys Arg His Gly LysLys Ser Arg Leu Arg Cys Ser Lys Lys Pro 10 15 20 Leu His Val Asn Phe LysGlu Leu Gly Trp Asp Asp Trp Ile Ile Ala 25 30 35 Pro Leu Glu Tyr Glu AlaTyr His Cys Glu Gly Val Cys Asp Phe Pro 40 45 50 55 Leu Arg Ser His LeuGlu Pro Thr Asn His Ala Ile Ile Gln Thr Leu 60 65 70 Met Asn Ser Met AspPro Gly Ser Thr Pro Pro Ser Cys Cys Val Pro 75 80 85 Thr Lys Leu Thr ProIle Ser Ile Leu Tyr Ile Asp Ala Gly Asn Asn 90 95 100 Val Val Tyr LysGln Tyr Glu Asp Met Val Val Glu Ser Cys Gly Cys 105 110 115 Arg 120 1233base pairs nucleic acid single linear DNA (genomic) not provided DNAencoding BMP2 propeptide/BMP-12 mature CDS 1..1233 mat_peptide 847..123327 ATG GTG GCC GGG ACC CGC TGT CTT CTA GCG TTG CTG CTT CCC CAG GTC 48Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val -282-280 -275 -270 CTC CTG GGC GGC GCG GCT GGC CTC GTT CCG GAG CTG GGC CGCAGG AAG 96 Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg ArgLys -265 -260 -255 TTC GCG GCG GCG TCG TCG GGC CGC CCC TCA TCC CAG CCCTCT GAC GAG 144 Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser Gln Pro SerAsp Glu -250 -245 -240 -235 GTC CTG AGC GAG TTC GAG TTG CGG CTG CTC AGCATG TTC GGC CTG AAA 192 Val Leu Ser Glu Phe Glu Leu Arg Leu Leu Ser MetPhe Gly Leu Lys -230 -225 -220 CAG AGA CCC ACC CCC AGC AGG GAC GCC GTGGTG CCC CCC TAC ATG CTA 240 Gln Arg Pro Thr Pro Ser Arg Asp Ala Val ValPro Pro Tyr Met Leu -215 -210 -205 GAC CTG TAT CGC AGG CAC TCA GGT CAGCCG GGC TCA CCC GCC CCA GAC 288 Asp Leu Tyr Arg Arg His Ser Gly Gln ProGly Ser Pro Ala Pro Asp -200 -195 -190 CAC CGG TTG GAG AGG GCA GCC AGCCGA GCC AAC ACT GTG CGC AGC TTC 336 His Arg Leu Glu Arg Ala Ala Ser ArgAla Asn Thr Val Arg Ser Phe -185 -180 -175 CAC CAT GAA GAA TCT TTG GAAGAA CTA CCA GAA ACG AGT GGG AAA ACA 384 His His Glu Glu Ser Leu Glu GluLeu Pro Glu Thr Ser Gly Lys Thr -170 -165 -160 -155 ACC CGG AGA TTC TTCTTT AAT TTA AGT TCT ATC CCC ACG GAG GAG TTT 432 Thr Arg Arg Phe Phe PheAsn Leu Ser Ser Ile Pro Thr Glu Glu Phe -150 -145 -140 ATC ACC TCA GCAGAG CTT CAG GTT TTC CGA GAA CAG ATG CAA GAT GCT 480 Ile Thr Ser Ala GluLeu Gln Val Phe Arg Glu Gln Met Gln Asp Ala -135 -130 -125 TTA GGA AACAAT AGC AGT TTC CAT CAC CGA ATT AAT ATT TAT GAA ATC 528 Leu Gly Asn AsnSer Ser Phe His His Arg Ile Asn Ile Tyr Glu Ile -120 -115 -110 ATA AAACCT GCA ACA GCC AAC TCG AAA TTC CCC GTG ACC AGA CTT TTG 576 Ile Lys ProAla Thr Ala Asn Ser Lys Phe Pro Val Thr Arg Leu Leu -105 -100 -95 GACACC AGG TTG GTG AAT CAG AAT GCA AGC AGG TGG GAA AGT TTT GAT 624 Asp ThrArg Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp -90 -85 -80 -75GTC ACC CCC GCT GTG ATG CGG TGG ACT GCA CAG GGA CAC GCC AAC CAT 672 ValThr Pro Ala Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His -70 -65 -60GGA TTC GTG GTG GAA GTG GCC CAC TTG GAG GAG AAA CAA GGT GTC TCC 720 GlyPhe Val Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser -55 -50 -45AAG AGA CAT GTT AGG ATA AGC AGG TCT TTG CAC CAA GAT GAA CAC AGC 768 LysArg His Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser -40 -35 -30TGG TCA CAG ATA AGG CCA TTG CTA GTA ACT TTT GGC CAT GAT GGA AAA 816 TrpSer Gln Ile Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys -25 -20 -15GGG CAT CCT CTC CAC AAA AGA GAA AAA CGT ACG GCG TTG GCC GGG ACG 864 GlyHis Pro Leu His Lys Arg Glu Lys Arg Thr Ala Leu Ala Gly Thr -10 -5 1 5CGG ACA GCG CAG GGC AGC GGC GGG GGC GCG GGC CGG GGC CAC GGG CGC 912 ArgThr Ala Gln Gly Ser Gly Gly Gly Ala Gly Arg Gly His Gly Arg 10 15 20 AGGGGC CGG AGC CGC TGC AGC CGC AAG CCG TTG CAC GTG GAC TTC AAG 960 Arg GlyArg Ser Arg Cys Ser Arg Lys Pro Leu His Val Asp Phe Lys 25 30 35 GAG CTCGGC TGG GAC GAC TGG ATC ATC GCG CCG CTG GAC TAC GAG GCG 1008 Glu Leu GlyTrp Asp Asp Trp Ile Ile Ala Pro Leu Asp Tyr Glu Ala 40 45 50 TAC CAC TGCGAG GGC CTT TGC GAC TTC CCT TTG CGT TCG CAC CTC GAG 1056 Tyr His Cys GluGly Leu Cys Asp Phe Pro Leu Arg Ser His Leu Glu 55 60 65 70 CCC ACC AACCAT GCC ATC ATT CAG ACG CTG CTC AAC TCC ATG GCA CCA 1104 Pro Thr Asn HisAla Ile Ile Gln Thr Leu Leu Asn Ser Met Ala Pro 75 80 85 GAC GCG GCG CCGGCC TCC TGC TGT GTG CCA GCG CGC CTC AGC CCC ATC 1152 Asp Ala Ala Pro AlaSer Cys Cys Val Pro Ala Arg Leu Ser Pro Ile 90 95 100 AGC ATC CTC TACATC GAC GCC GCC AAC AAC GTT GTC TAC AAG CAA TAC 1200 Ser Ile Leu Tyr IleAsp Ala Ala Asn Asn Val Val Tyr Lys Gln Tyr 105 110 115 GAG GAC ATG GTGGTG GAG GCC TGC GGC TGC AGG 1233 Glu Asp Met Val Val Glu Ala Cys Gly CysArg 120 125 411 amino acids amino acid linear protein not provided 28Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val -282-280 -275 -270 Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly ArgArg Lys -265 -260 -255 Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser GlnPro Ser Asp Glu -250 -245 -240 -235 Val Leu Ser Glu Phe Glu Leu Arg LeuLeu Ser Met Phe Gly Leu Lys -230 -225 -220 Gln Arg Pro Thr Pro Ser ArgAsp Ala Val Val Pro Pro Tyr Met Leu -215 -210 -205 Asp Leu Tyr Arg ArgHis Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp -200 -195 -190 His Arg LeuGlu Arg Ala Ala Ser Arg Ala Asn Thr Val Arg Ser Phe -185 -180 -175 HisHis Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr -170 -165-160 -155 Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu GluPhe -150 -145 -140 Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln MetGln Asp Ala -135 -130 -125 Leu Gly Asn Asn Ser Ser Phe His His Arg IleAsn Ile Tyr Glu Ile -120 -115 -110 Ile Lys Pro Ala Thr Ala Asn Ser LysPhe Pro Val Thr Arg Leu Leu -105 -100 -95 Asp Thr Arg Leu Val Asn GlnAsn Ala Ser Arg Trp Glu Ser Phe Asp -90 -85 -80 -75 Val Thr Pro Ala ValMet Arg Trp Thr Ala Gln Gly His Ala Asn His -70 -65 -60 Gly Phe Val ValGlu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser -55 -50 -45 Lys Arg HisVal Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser -40 -35 -30 Trp SerGln Ile Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys -25 -20 -15 GlyHis Pro Leu His Lys Arg Glu Lys Arg Thr Ala Leu Ala Gly Thr -10 -5 1 5Arg Thr Ala Gln Gly Ser Gly Gly Gly Ala Gly Arg Gly His Gly Arg 10 15 20Arg Gly Arg Ser Arg Cys Ser Arg Lys Pro Leu His Val Asp Phe Lys 25 30 35Glu Leu Gly Trp Asp Asp Trp Ile Ile Ala Pro Leu Asp Tyr Glu Ala 40 45 50Tyr His Cys Glu Gly Leu Cys Asp Phe Pro Leu Arg Ser His Leu Glu 55 60 6570 Pro Thr Asn His Ala Ile Ile Gln Thr Leu Leu Asn Ser Met Ala Pro 75 8085 Asp Ala Ala Pro Ala Ser Cys Cys Val Pro Ala Arg Leu Ser Pro Ile 90 95100 Ser Ile Leu Tyr Ile Asp Ala Ala Asn Asn Val Val Tyr Lys Gln Tyr 105110 115 Glu Asp Met Val Val Glu Ala Cys Gly Cys Arg 120 125 1203 basepairs nucleic acid single linear DNA (genomic) not provided murine MV1CDS 2..721 29 A AAG TTC TGC CTG GTG CTG GNG NCG GTG ACG GCC TCG GAG AGCAGN 46 Lys Phe Cys Leu Val Leu Xaa Xaa Val Thr Ala Ser Glu Ser Xaa 1 510 15 CNG CTG GCC CTG AGA CGA CTG GGC TTC GGC TGN CCG GGC GGT GGC GAC 94Xaa Leu Ala Leu Arg Arg Leu Gly Phe Gly Xaa Pro Gly Gly Gly Asp 20 25 30GGC GGC GGC ACT GCG GNC GAG GAG CGC GCG CTG TTG GTG ATC TCC TCC 142 GlyGly Gly Thr Ala Xaa Glu Glu Arg Ala Leu Leu Val Ile Ser Ser 35 40 45 CGTACG CAA AGG AAA GAG AGT CTG TTC CGG GAG ATC CGA GCC CAG GCC 190 Arg ThrGln Arg Lys Glu Ser Leu Phe Arg Glu Ile Arg Ala Gln Ala 50 55 60 CGT GCTCTC CGG GCC GCT GCA GAG CCG CCA CCG GAT CCA GGA CCA GGC 238 Arg Ala LeuArg Ala Ala Ala Glu Pro Pro Pro Asp Pro Gly Pro Gly 65 70 75 GCT GGG TCACGC AAA GCC AAC CTG GGC GGT CGC AGG CGG CAG CGG ACT 286 Ala Gly Ser ArgLys Ala Asn Leu Gly Gly Arg Arg Arg Gln Arg Thr 80 85 90 95 GCG CTG GCTGGG ACT CGG GGA GNG NAG GGA AGC GGT GGT GGC GGC GGT 334 Ala Leu Ala GlyThr Arg Gly Xaa Xaa Gly Ser Gly Gly Gly Gly Gly 100 105 110 GGC GGT GGCGGC GGC GGC GGC GGC GGC GGC GGC GGC GGC GGC GGC GCA 382 Gly Gly Gly GlyGly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala 115 120 125 GGC AGG GGCCAC GGG CGC AGA GGC CGG AGC CGC TGC GGT CGC AAG TCA 430 Gly Arg Gly HisGly Arg Arg Gly Arg Ser Arg Cys Gly Arg Lys Ser 130 135 140 CTG CAC GTGGAC TTT AAG GAG CTG GGC TGG GAC GAC TGG ATC ATC GCG 478 Leu His Val AspPhe Lys Glu Leu Gly Trp Asp Asp Trp Ile Ile Ala 145 150 155 CCA TTA GACTAC GAG GCA TAC CAC TGC GAG GGC GTT TGC GAC TTT CCT 526 Pro Leu Asp TyrGlu Ala Tyr His Cys Glu Gly Val Cys Asp Phe Pro 160 165 170 175 CTG CGCTCG CAC CTG GAG CCT ACC AAC CAC GCC ATC ATT CAG ACG CTG 574 Leu Arg SerHis Leu Glu Pro Thr Asn His Ala Ile Ile Gln Thr Leu 180 185 190 CTC AACTCC ATG GCG CCC GAC GCT GCG CCA GCC TCC TGC TGC GTG CCC 622 Leu Asn SerMet Ala Pro Asp Ala Ala Pro Ala Ser Cys Cys Val Pro 195 200 205 GCA AGGCTC AGT CCC ATC AGC ATT CTC TAC ATC GAT GCC GCC AAC AAC 670 Ala Arg LeuSer Pro Ile Ser Ile Leu Tyr Ile Asp Ala Ala Asn Asn 210 215 220 GTG GTCTAC AAG CAG TAC GAA GAC ATG GTG GTG GAG GCC TGC GGC TGC 718 Val Val TyrLys Gln Tyr Glu Asp Met Val Val Glu Ala Cys Gly Cys 225 230 235 AGGTAGCATGCGG TCTGGGGAGG GTCTGGCCGC CCAGGACCCT AGCTCAAGAG 771 Arg 240CAGGTGTCAT CAGGCCCGAG GGACGGCGGA CTATGGCCTC TGCCAGCACA GAGGAGAGCA 831CACAGTTAAC ACTCACATTT ACACACTCCT TCACTCACGC ACATGTTTAC CGTGGACGGC 891AGGCGCTAAA AGCCTTGCTT ATTTGCTACC ATTGATACAA ACCTCTGTCC TTTTCGGGAG 951AGGGAAGGGC ATCTGTGTTT ATGTTGCAGT AATTGGCACT AAATCCAAGT AGAAATGGGT 1011TAGCATTGGA TTCTCCTTTT AGTTGGAGGC GGTGTGGCTG GATTCCTGAC GTTGGATATG 1071GAGTGCACTG CAGGGCTGGG ATACCCAGAT TCTCTGGAGT GGGCATTGGG AACCTTCAAA 1131AGTAAGGAGC CACTGGGGCT TGGGAGGGAG CACCCGGTTC CTAAACAAGT CTGATGTGTA 1191CTGCTCAGTT TG 1203 240 amino acids amino acid linear protein notprovided 30 Lys Phe Cys Leu Val Leu Xaa Xaa Val Thr Ala Ser Glu Ser XaaXaa 1 5 10 15 Leu Ala Leu Arg Arg Leu Gly Phe Gly Xaa Pro Gly Gly GlyAsp Gly 20 25 30 Gly Gly Thr Ala Xaa Glu Glu Arg Ala Leu Leu Val Ile SerSer Arg 35 40 45 Thr Gln Arg Lys Glu Ser Leu Phe Arg Glu Ile Arg Ala GlnAla Arg 50 55 60 Ala Leu Arg Ala Ala Ala Glu Pro Pro Pro Asp Pro Gly ProGly Ala 65 70 75 80 Gly Ser Arg Lys Ala Asn Leu Gly Gly Arg Arg Arg GlnArg Thr Ala 85 90 95 Leu Ala Gly Thr Arg Gly Xaa Xaa Gly Ser Gly Gly GlyGly Gly Gly 100 105 110 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Ala Gly 115 120 125 Arg Gly His Gly Arg Arg Gly Arg Ser Arg CysGly Arg Lys Ser Leu 130 135 140 His Val Asp Phe Lys Glu Leu Gly Trp AspAsp Trp Ile Ile Ala Pro 145 150 155 160 Leu Asp Tyr Glu Ala Tyr His CysGlu Gly Val Cys Asp Phe Pro Leu 165 170 175 Arg Ser His Leu Glu Pro ThrAsn His Ala Ile Ile Gln Thr Leu Leu 180 185 190 Asn Ser Met Ala Pro AspAla Ala Pro Ala Ser Cys Cys Val Pro Ala 195 200 205 Arg Leu Ser Pro IleSer Ile Leu Tyr Ile Asp Ala Ala Asn Asn Val 210 215 220 Val Tyr Lys GlnTyr Glu Asp Met Val Val Glu Ala Cys Gly Cys Arg 225 230 235 240 1046base pairs nucleic acid single linear DNA (genomic) NO NO not providedMURINE MV2 CDS 2..790 31 A AGA AAA CAA GCT TGC ATT CCT GCA GGT CCG ACTCTA AGA GGA TCC 46 Arg Lys Gln Ala Cys Ile Pro Ala Gly Pro Thr Leu ArgGly Ser 1 5 10 15 TCA GGG ACC CAA CCC AGG CCG GCT GGG AAG TCT TTC GACGTG TGG CAG 94 Ser Gly Thr Gln Pro Arg Pro Ala Gly Lys Ser Phe Asp ValTrp Gln 20 25 30 GGC CTG CGC CCT CAG CCT TGG AAG CAG CTG TGC CTG GAG TTGCGG GCA 142 Gly Leu Arg Pro Gln Pro Trp Lys Gln Leu Cys Leu Glu Leu ArgAla 35 40 45 GCC TGG GGT GAG CTG GAC RCC GGG GAT ACG GGG GCG CGC GCG AGGGGT 190 Ala Trp Gly Glu Leu Asp Xaa Gly Asp Thr Gly Ala Arg Ala Arg Gly50 55 60 CCC CAG CAG CCA CCG CCT CTG GAC CTG CGG AGT CTG GGC TTC GGT CGG238 Pro Gln Gln Pro Pro Pro Leu Asp Leu Arg Ser Leu Gly Phe Gly Arg 6570 75 AGG GTG AGA CCG CCC CAG GAG CGC GCC CTG CTT GTA GTG TTC ACC AGA286 Arg Val Arg Pro Pro Gln Glu Arg Ala Leu Leu Val Val Phe Thr Arg 8085 90 95 TCG CAG CGC AAG AAC CTG TTC ACT GAG ATG CAT GAG CAG CTG GGC TCT334 Ser Gln Arg Lys Asn Leu Phe Thr Glu Met His Glu Gln Leu Gly Ser 100105 110 GCA GAG GCT GCG GGA GCC GAG GGG TCA TGT CCA GCG CCG TCG GGC TCC382 Ala Glu Ala Ala Gly Ala Glu Gly Ser Cys Pro Ala Pro Ser Gly Ser 115120 125 CCA GAC ACC GGG TCT TGG CTG CCC TCG CCC GGC CGC CGG CGG CGA CGC430 Pro Asp Thr Gly Ser Trp Leu Pro Ser Pro Gly Arg Arg Arg Arg Arg 130135 140 ACC GCC TTC GCC AGC CGT CAC GGC AAG CGA CAT GGC AAG AAG TCC AGG478 Thr Ala Phe Ala Ser Arg His Gly Lys Arg His Gly Lys Lys Ser Arg 145150 155 CTG CGC TGC AGC AGA AAG CCT CTG CAC GTG AAT TTT AAG GAG TTA GGC526 Leu Arg Cys Ser Arg Lys Pro Leu His Val Asn Phe Lys Glu Leu Gly 160165 170 175 TGG GAC GAC TGG ATT ATC GCG CCC CTA GAG TAC GAG GCC TAT CACTGC 574 Trp Asp Asp Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Tyr His Cys180 185 190 GAG GGC GTG TGC GAC TTT CCG CTG CGC TCG CAC CTT GAG CCC ACTAAC 622 Glu Gly Val Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn195 200 205 CAT GCC ATC ATT CAG ACG CTG ATG AAC TCC ATG GAC CCG GGC TCCACC 670 His Ala Ile Ile Gln Thr Leu Met Asn Ser Met Asp Pro Gly Ser Thr210 215 220 CCG CCT AGC TGC TGC GTT CCC ACC AAA CTG ACT CCC ATT AGC ATCCTG 718 Pro Pro Ser Cys Cys Val Pro Thr Lys Leu Thr Pro Ile Ser Ile Leu225 230 235 TAC ATC GAC GCG GGC AAT AAT GTN GTC TAC AAG CAG TAT GAG GACATG 766 Tyr Ile Asp Ala Gly Asn Asn Xaa Val Tyr Lys Gln Tyr Glu Asp Met240 245 250 255 GTG GTG GAG TCC TGC GGC TGT AGG TAGCGGTGCT GTCCCGCCACCTGGGCCAGG 820 Val Val Glu Ser Cys Gly Cys Arg 260 GACCATGGAG GGAGGCCTGACTGCCGAGAA AGGAGCAGGA GCTGGCCTTG GAAGAGGCCA 880 CAGGTGGGGG ACAGCCTGAAAGTAGGAGCA CAGTAAGAAG CAGCCCAGCC TTCCCAGAAC 940 CTTCCAATCC CCCAACCCAGAAGCAGCTAA GGGGTTTCAC AACTTTTGGC CTTGCCAGCC 1000 TGGAAAGACT AGACAAGAGGGATTCTTCTC TTTTTATTAT GGCTTG 1046 263 amino acids amino acid linearprotein not provided 32 Arg Lys Gln Ala Cys Ile Pro Ala Gly Pro Thr LeuArg Gly Ser Ser 1 5 10 15 Gly Thr Gln Pro Arg Pro Ala Gly Lys Ser PheAsp Val Trp Gln Gly 20 25 30 Leu Arg Pro Gln Pro Trp Lys Gln Leu Cys LeuGlu Leu Arg Ala Ala 35 40 45 Trp Gly Glu Leu Asp Xaa Gly Asp Thr Gly AlaArg Ala Arg Gly Pro 50 55 60 Gln Gln Pro Pro Pro Leu Asp Leu Arg Ser LeuGly Phe Gly Arg Arg 65 70 75 80 Val Arg Pro Pro Gln Glu Arg Ala Leu LeuVal Val Phe Thr Arg Ser 85 90 95 Gln Arg Lys Asn Leu Phe Thr Glu Met HisGlu Gln Leu Gly Ser Ala 100 105 110 Glu Ala Ala Gly Ala Glu Gly Ser CysPro Ala Pro Ser Gly Ser Pro 115 120 125 Asp Thr Gly Ser Trp Leu Pro SerPro Gly Arg Arg Arg Arg Arg Thr 130 135 140 Ala Phe Ala Ser Arg His GlyLys Arg His Gly Lys Lys Ser Arg Leu 145 150 155 160 Arg Cys Ser Arg LysPro Leu His Val Asn Phe Lys Glu Leu Gly Trp 165 170 175 Asp Asp Trp IleIle Ala Pro Leu Glu Tyr Glu Ala Tyr His Cys Glu 180 185 190 Gly Val CysAsp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His 195 200 205 Ala IleIle Gln Thr Leu Met Asn Ser Met Asp Pro Gly Ser Thr Pro 210 215 220 ProSer Cys Cys Val Pro Thr Lys Leu Thr Pro Ile Ser Ile Leu Tyr 225 230 235240 Ile Asp Ala Gly Asn Asn Xaa Val Tyr Lys Gln Tyr Glu Asp Met Val 245250 255 Val Glu Ser Cys Gly Cys Arg 260 1345 base pairs nucleic acidsingle linear DNA (genomic) NO NO not provided HUMAN V1-1 CDS 138..1301mat_peptide 990..1301 33 AACTATAGCA CCTGCAGTCC CTGGTCTTGG GTGTAGGGGTGCGCTCCTGG TCCCGCGGCT 60 CAGGGATATG CAGTGACCAA TGGGTTGTTG GCCTGATGGGACTTTTGGCT TGCTAAACCA 120 AAGCTCGGTT CGGATAG CCC GGG CGA AGA CGT CCG CTGCTC TGG GCC AGG 170 Pro Gly Arg Arg Arg Pro Leu Leu Trp Ala Arg -284-280 -275 CTG GCA GCG TTC AGG CTG GGG CAG AGA CGC GGA GTC GGG CGC TGGCTC 218 Leu Ala Ala Phe Arg Leu Gly Gln Arg Arg Gly Val Gly Arg Trp Leu-270 -265 -260 CAA CAG GCC TGG CTC CCA CAT CGA AGA CAG CTG GGC CAT TTGCTG TTA 266 Gln Gln Ala Trp Leu Pro His Arg Arg Gln Leu Gly His Leu LeuLeu -255 -250 -245 GGA GGC CCC GCG CTG ACA GTG TGC AGG ATT TGC TCT TACACA GCT CTT 314 Gly Gly Pro Ala Leu Thr Val Cys Arg Ile Cys Ser Tyr ThrAla Leu -240 -235 -230 TCT CTC TGT CCC TGC CGG TCC CCC GCA GAC GAA TCGGCA GCC GAA ACA 362 Ser Leu Cys Pro Cys Arg Ser Pro Ala Asp Glu Ser AlaAla Glu Thr -225 -220 -215 -210 GGC CAG AGC TTC CTG TTC GAC GTG TCC AGCCTT AAC GAC GCA GAC GAG 410 Gly Gln Ser Phe Leu Phe Asp Val Ser Ser LeuAsn Asp Ala Asp Glu -205 -200 -195 GTG GTG GGT GCC GAG CTG CGC GTG CTGCGC CGG GGA TCT CCA GAG TCG 458 Val Val Gly Ala Glu Leu Arg Val Leu ArgArg Gly Ser Pro Glu Ser -190 -185 -180 GGC CCA GGC AGC TGG ACT TCT CCGCCG TTG CTG CTG CTG TCC ACG TGC 506 Gly Pro Gly Ser Trp Thr Ser Pro ProLeu Leu Leu Leu Ser Thr Cys -175 -170 -165 CCG GGC GCC GCC CGA GCG CCACGC CTG CTG TAC TCG CGG GCA GCT GAG 554 Pro Gly Ala Ala Arg Ala Pro ArgLeu Leu Tyr Ser Arg Ala Ala Glu -160 -155 -150 CCC CTA GTC GGT CAG CGCTGG GAG GCG TTC GAC GTG GCG GAC GCC ATG 602 Pro Leu Val Gly Gln Arg TrpGlu Ala Phe Asp Val Ala Asp Ala Met -145 -140 -135 -130 AGG CGC CAC CGTCGT GAA CCG CGC CCC CCC CGC GCG TTC TGC CTC TTG 650 Arg Arg His Arg ArgGlu Pro Arg Pro Pro Arg Ala Phe Cys Leu Leu -125 -120 -115 CTG CGC GCAGTG GCA GGC CCG GTG CCG AGC CCG TTG GCA CTG CGG CGA 698 Leu Arg Ala ValAla Gly Pro Val Pro Ser Pro Leu Ala Leu Arg Arg -110 -105 -100 CTG GGCTTC GGC TGG CCG GGC GGA GGG GGC TCT GCG GCA GAG GAG CGC 746 Leu Gly PheGly Trp Pro Gly Gly Gly Gly Ser Ala Ala Glu Glu Arg -95 -90 -85 GCG GTGCTA GTC GTC TCC TCC CGC ACG CAG AGG AAA GAG AGC TTA TTC 794 Ala Val LeuVal Val Ser Ser Arg Thr Gln Arg Lys Glu Ser Leu Phe -80 -75 -70 CGG GAGATC CGC GCC CAG GCC CGC GCG CTC GGG GCC GCT CTG GCC TCA 842 Arg Glu IleArg Ala Gln Ala Arg Ala Leu Gly Ala Ala Leu Ala Ser -65 -60 -55 -50 GAGCCG CTG CCC GAC CCA GGA ACC GGC ACC GCG TCG CCA AGG GCA GTC 890 Glu ProLeu Pro Asp Pro Gly Thr Gly Thr Ala Ser Pro Arg Ala Val -45 -40 -35 ATTGGC GGC CGC AGA CGG AGG AGG ACG GCG TTG GCC GGG ACG CGG ACA 938 Ile GlyGly Arg Arg Arg Arg Arg Thr Ala Leu Ala Gly Thr Arg Thr -30 -25 -20 GCGCAG GGC AGC GGC GGG GGC GCG GGC CGG GGC CAC GGG CGC AGG GGC 986 Ala GlnGly Ser Gly Gly Gly Ala Gly Arg Gly His Gly Arg Arg Gly -15 -10 -5 CGGAGC CGC TGC AGC CGC AAG CCG TTG CAC GTG GAC TTC AAG GAG CTC 1034 Arg SerArg Cys Ser Arg Lys Pro Leu His Val Asp Phe Lys Glu Leu 1 5 10 15 GGCTGG GAC GAC TGG ATC ATC GCG CCG CTG GAC TAC GAG GCG TAC CAC 1082 Gly TrpAsp Asp Trp Ile Ile Ala Pro Leu Asp Tyr Glu Ala Tyr His 20 25 30 TGC GAGGGC CTT TGC GAC TTC CCT TTG CGT TCG CAC CTC GAG CCC ACC 1130 Cys Glu GlyLeu Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr 35 40 45 AAC CAT GCCATC ATT CAG ACG CTG CTC AAC TCC ATG GCA CCA GAC GCG 1178 Asn His Ala IleIle Gln Thr Leu Leu Asn Ser Met Ala Pro Asp Ala 50 55 60 GCG CCG GCC TCCTGC TGT GTG CCA GCG CGC CTC AGC CCC ATC AGC ATC 1226 Ala Pro Ala Ser CysCys Val Pro Ala Arg Leu Ser Pro Ile Ser Ile 65 70 75 CTC TAC ATC GAC GCCGCC AAC AAC GTT GTC TAC AAG CAA TAC GAG GAC 1274 Leu Tyr Ile Asp Ala AlaAsn Asn Val Val Tyr Lys Gln Tyr Glu Asp 80 85 90 95 ATG GTG GTG GAG GCCTGC GGC TGC AGG TAGCGCGCGG GCCGGGGAGG 1321 Met Val Val Glu Ala Cys GlyCys Arg 100 GGGCAGCCAC GCGGCCGAGG ATCC 1345 388 amino acids amino acidlinear protein not provided 34 Pro Gly Arg Arg Arg Pro Leu Leu Trp AlaArg Leu Ala Ala Phe Arg -284 -280 -275 -270 Leu Gly Gln Arg Arg Gly ValGly Arg Trp Leu Gln Gln Ala Trp Leu -265 -260 -255 Pro His Arg Arg GlnLeu Gly His Leu Leu Leu Gly Gly Pro Ala Leu -250 -245 -240 Thr Val CysArg Ile Cys Ser Tyr Thr Ala Leu Ser Leu Cys Pro Cys -235 -230 -225 ArgSer Pro Ala Asp Glu Ser Ala Ala Glu Thr Gly Gln Ser Phe Leu -220 -215-210 -205 Phe Asp Val Ser Ser Leu Asn Asp Ala Asp Glu Val Val Gly AlaGlu -200 -195 -190 Leu Arg Val Leu Arg Arg Gly Ser Pro Glu Ser Gly ProGly Ser Trp -185 -180 -175 Thr Ser Pro Pro Leu Leu Leu Leu Ser Thr CysPro Gly Ala Ala Arg -170 -165 -160 Ala Pro Arg Leu Leu Tyr Ser Arg AlaAla Glu Pro Leu Val Gly Gln -155 -150 -145 Arg Trp Glu Ala Phe Asp ValAla Asp Ala Met Arg Arg His Arg Arg -140 -135 -130 -125 Glu Pro Arg ProPro Arg Ala Phe Cys Leu Leu Leu Arg Ala Val Ala -120 -115 -110 Gly ProVal Pro Ser Pro Leu Ala Leu Arg Arg Leu Gly Phe Gly Trp -105 -100 -95Pro Gly Gly Gly Gly Ser Ala Ala Glu Glu Arg Ala Val Leu Val Val -90 -85-80 Ser Ser Arg Thr Gln Arg Lys Glu Ser Leu Phe Arg Glu Ile Arg Ala -75-70 -65 Gln Ala Arg Ala Leu Gly Ala Ala Leu Ala Ser Glu Pro Leu Pro Asp-60 -55 -50 -45 Pro Gly Thr Gly Thr Ala Ser Pro Arg Ala Val Ile Gly GlyArg Arg -40 -35 -30 Arg Arg Arg Thr Ala Leu Ala Gly Thr Arg Thr Ala GlnGly Ser Gly -25 -20 -15 Gly Gly Ala Gly Arg Gly His Gly Arg Arg Gly ArgSer Arg Cys Ser -10 -5 1 Arg Lys Pro Leu His Val Asp Phe Lys Glu Leu GlyTrp Asp Asp Trp 5 10 15 20 Ile Ile Ala Pro Leu Asp Tyr Glu Ala Tyr HisCys Glu Gly Leu Cys 25 30 35 Asp Phe Pro Leu Arg Ser His Leu Glu Pro ThrAsn His Ala Ile Ile 40 45 50 Gln Thr Leu Leu Asn Ser Met Ala Pro Asp AlaAla Pro Ala Ser Cys 55 60 65 Cys Val Pro Ala Arg Leu Ser Pro Ile Ser IleLeu Tyr Ile Asp Ala 70 75 80 Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu AspMet Val Val Glu Ala 85 90 95 100 Cys Gly Cys Arg 17 base pairs nucleicacid single linear DNA (genomic) NO NO not provided primer number 8 35TGTATGCGAC TTCCCGC 17

What is claimed is:
 1. A chimeric DNA molecule comprising a DNA sequenceencoding a propeptide from a member of the TGF-β superfamily of proteinslinked in correct reading from to a DNA sequence encoding an amino acidsequence encoding a mature polypeptide, said polypeptide selected fromthe group consisting of Bone Morphogenetic Protein-12, BMP-13 and MP52polypeptides.
 2. A chimeric DNA molecule according to claim 1, whereinthe propeptide is the propeptide from BMP-2.
 3. A heterodimericpolypeptide molecule comprising one monomer having the amino acidsequence selected from the group consisting of BMP-12, BMP-13 and MP52polypeptides, and one monomer having the amino acid sequence of apolypeptide of the TGF-β superfamily.
 4. A heterodimeric polypeptidemolecule according to claim 3, wherein one monomer comprises the aminoacid sequence of BMP-12, and one monomer comprises the amino acidsequence of a protein selected from the group consisting of BMP-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP7, BMP-8, BMP-9, BMP-10 and BMP-11.
 5. Aheterodimeric polypeptide molecule wherein each monomer comprises anindependently selected amino acid sequence encoding a polypeptideselected from BMP-12, BMP-13 and MP-52.